DNA encoding mammalian phosphodiesterases

ABSTRACT

The present invention relates to novel purified and isolated nucleotide sequences encoding mammalian Ca 2+ /calmodulin stimulated phosphodiesterases (CaM-PDEs) and cyclic-GMP-stimulated phosphodiesterases (cGS-PDEs). Also provided are the corresponding recombinant expression products of said nucleotide sequences, immunological reagents specifically reactive therewith, and procedures for identifying compounds which modulate the enzymatic activity of such expression products.

This is a continuation-in-part of our co-pending U.S. patent applicationSer. No. 07/688,356, filed Apr. 19, 1991.

BACKGROUND OF THE INVENTION

The present invention relates to novel purified and isolated nucleotidesequences encoding mammalian Ca²⁺/calmodulin stimulatedphosphodiesterases (CaM-PDEs) and cyclic-GMP-stimulatedphosphodiesterases (cGS-PDEs). Also provided are the correspondingrecombinant expression products of said nucleotide sequences,immunological reagents specifically reactive therewith, and proceduresfor identifying compounds which modulate the enzymatic activity of suchexpression products.

Cyclic nucleotides are known to mediate a wide variety of cellularresponses to biological stimuli. The cyclic nucleotidephosphodiesterases (PDEs) catalyze the hydrolysis of 3′, 5′ cyclicnucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclicguanosine monophosphate (cGMP), to their corresponding 5′-nucleotidemonophosphates and are consequently important in the control of cellularconcentration of cyclic nucleotides. The PDEs in turn are regulated bytransmembrane signals or second messenger ligands such as calcium ion(Ca²⁺) or cGMP. The PDEs thus have a central role in regulating the flowof information from extracellular hormones, neurotransmitters, or othersignals that use the cyclic nucleotides as messengers.

PDEs are a large and complex group of enzymes. They are widelydistributed throughout the cells and tissues of most eukaryoticorganisms, but are usually present only in trace amounts. At least fivedifferent families of PDEs have been described based on characteristicssuch as substrate specificity, kinetic properties, cellular regulatory.control, size, and in some instances, modulation by selectiveinhibitors. [Beavo, Adv. in Second Mess. and Prot. Phosph. Res. 22:1-38(1988)]. The five families include:

-   -   I Ca²⁺/calmodulin-stimulated    -   II cGMP-stimulated    -   III cGMP-inhibited    -   IV cAMP-specific    -   V cGMP-specific

Within each family there are multiple forms of closely related PDEs. SeeBeavo, “Multiple Phosphodiesterase Isozymes Background, Nomenclature andImplications”, pp. 3-15; Wang et al., “Calmodulin-Stimulated CyclicNucleotide Phosphodiesterases”, pp. 19-59; and Manganiello et al.,“Cyclic GMP-Stimulated Cyclic Nucleotide Phosphodiesterases” pp. 62-85;all in Cyclic Nucleotide Phosphodiesterases: Structure, Regulation andDrua Action, Beavo, J. and Houslay, M. D., Eds.; John Wiley & Sons, NewYork (1990).

The Ca²⁺/calmodulin dependent PDEs (CaM-PDEs) are characterized by theirresponsiveness to intracellular calcium, which leads to a decreasedintracellulai concentration of cAMP and/or cGMP. A distinctive featureof cGMP-stimulated phosphodiesterases (cGS-PDEs) is their capacity to bestimulated by cGMP in effecting cAMP hydrolysis.

In vitro studies have shown increased PDE activity in response toCa²⁺/calmodulin in nearly every mammalian tissue studied, as well as inDrosophila, Dictyostelium, and trypanosomes. The level of CaM-PDE intissues and cellular and subcellular compartments varies widely. Mostcells contain at least a small amount of CaM-PDE activity, with thehighest tissue levels being found in the brain, particularly in thesynaptic areas. Greenberg et al. Neuropharmacol., 17:737-745 (1978) andKincaid et al., PNAS (USA), 84:1118-1122 (1987). A decrease in cAMP inastrocytoma cells in response to muscarinic stimulation may be due tocalcium dependent increases in CaM-PDE activity. Tanner et al., Mol.Pharmacol., 29:455-460 (1986). Also, CaM-PDE may be an importantregulator of cAMP in thyroid tissue. Erneux et al., Mol. Cell.Endocrinol., 43:123-134(1985).

Early studies suggested that there are distinct tissue-specific isozymesof CaM-PDEs. Several members of the CaM-PDE family have now beendescribed, including a 59 kDa isozyme isolated from bovine heart, and 61and 63 kDa isozymes isolated from bovine brain. LaPorte et al.,Biochemistry, 18:2820-2825 (1979); Hansen et al., Proc. Natl. Acad. Sci.USA, 79:2788-2792 (1982); and Sharma et al., J. Biol. Chem.,26:14160-14166 (1986). Possible counterparts to the bovine 59 and 61 kDaisozymes have also been isolated from rat tissues, Hansen et al., J.Biol. Chem., 261:14636-14645 (1986), suggesting that these two isozymesmay be expressed in other mammalian species.

In addition to molecular weight criteria, other evidence supports bothsimilarities and differences among the CaM=PDE family of isozymes. Forexample, the 59 kDa heart isozyme and the 61 kDa brain isozyme CaM-PDEsdiffer in mobility on SDS-PAGE and elution position on DEAEchromatography, and the 59 kDa isozyme has at least a 10-20 fold higheraffinity for calmodulin. Oncomodulin, a fetal/onco calcium bindingprotein present in very high concentrations in the placenta andtransformed cells, also binds to the 59 kDa enzyme with a higheraffinity than to the 61 kDa enzyme. However, both the 61 kDa brain andthe 59 kDa heart isozymes are recognized by a single monoclonalantibody. This antibody binds to the Ca²⁺/CaM-PDE complex with 100-foldhigher affinity than to PDE alone. Hansen et al., 1986, supra. The 59and 61 kDA isozymes have nearly identical substrate specificities andkinetic constants. Krinks et al., Adv. Cyc. Nucleotide Prot.Phosphorylation Res., 16:31-47 (1984) have suggested, based on peptidemapping experiments, that the heart 59 kDa protein could be aproteolytic form of the brain 61 kDa isozyme.

The 63 kDa bovine brain isozyme differs substantially from the 59 and 61kDa isozymes. The 63 kDa enzyme is not recognized by the monoclonalantibody which binds to the 59 and 61 kDa enzymes. Hansen et al., 1986,supra. The 63 kDa protein is not phosphorylated in vitro bycAMP-dependent protein kinase, whereas the 61 kDa protein isphosphorylated. Further, only the 63 kDa protein is phosphorylated invitro by CaM-kinase II. Sharma et al., Proc. Natl. Acad. Sci. (USA),82:2603-2607 (1985); and Hashimoto et al., J. Biol. Chem.,264:10884-10887 (1989). The 61 and 63 kDa CaM-PDE isozymes from bovinebrain do appear, however, to have similar CaM-binding affinities.Peptide maps generated by limited proteolysis with Staphylococcal V8protease, Sharma et al., J. Biol. Chem., 2:9248 (1984), have suggestedthat the 61 and 63 kDa proteins have different amino acid sequences.

The cGMP-stimulated PDEs (cGS-PDEs) are proposed to have a noncatalytic,cGMP-specific site that may account for the stimulation of cAMPhydrolysis by cGMP. Stoop et al., J. Biol. Chem., 2:13718 (1989). Atphysiological cyclic nucleotide concentrations, this enzyme responds toelevated cGMP concentrations with an enhanced hydrolysis of cAMP. Thus,cGS-PDE allows for increases in cGMP concentration to moderate orinhibit cAMP-mediated responses. The primary sequence presented recentlyin LeTrong et al., Biochemistry, 29:10280 (1990), co-authored by theinventors herein, provides the molecular framework for understanding theregulatory properties and domain substructure of this enzyme and forcomparing it with other PDE isozymes that respond to different signals.This publication also notes the cloning of a 2.2 kb bovine adrenalcortex cDNA fragment encoding cGS-PDE. See also, Thompson et al., FASEBJ., 5(6):A1592 (Abstract No. 7092) reporting on the cloning of a “TypeII PDE” from rat pheochromocytoma cells.

With the discovery of the large number of different PDEs and theircritical role in intracellular signalling, efforts have focused onfinding agents that selectively activate or inhibit specific PDEisozymes. Agents which affect cellular PDE activity, and thus altercellular cAMP, can potentially be used to control a broad range ofdiseases and physiological conditions. Some drugs which raise cAMPlevels by inhibiting PDEs are in use, but generally act as broadnonspecific inhibitors and have deleterious side effects on cAMPactivity in nontargeted tissues and cell types. Accordingly, agents areneeded which are specific for selected PDE isozymes. Selectiveinhibitors of specific PDE isozymes may be useful as cardiotonic agents,anti-depressants, anti-hypertensives, anti-thrombotics, and as otheragents. Screening studies for agonists/antagonists have beencomplicated, however, because of difficulties in identifying theparticular PDE isozyme present in a particular assay preparation.Moreover, all PDEs catalyze the same basic reaction; all haveoverlapping substrate specificities; and all occur only in traceamounts.

Differentiating among PDEs has been attempted by several differentmeans. The classical enzymological approach of isolating and studyingeach new isozyme is hampered by current limits of purificationtechniques and by the inability to accurately assess whether completeresolution of an isozyme has been achieved. A second approach has beento identify isozyme-specific assay conditions which might favor thecontribution of one isozyme and minimize that of others. Anotherapproach has been the immunological identification and separation intofamily groups and/or individual isozymes. There are obvious problemswith each of these approaches; for the unambiguous identification andstudy of a particular isozyme, a large number of distinguishing criterianeed to be established, which is often time consuming and in some casestechnically quite difficult. As a result, most studies have been donewith only partially pure PDE preparations that probably contained morethan one isozyme. Moreover, many of the PDEs in most tissues are verysusceptible to limited proteolysis and easily form active proteolyticproducts that may have different kinetic, regulatory, and physiologicalproperties from their parent form.

The development of new and specific PDE-modulatory agents would begreatly facilitated by the ability to isolate large quantities oftissue-specific PDEs by recombinant means. Relatively few PDE genes havebeen cloned to date and of those cloned, most belong to the cAM-specificfamily of phosphodiesterases (cAMP-PDEs). See Davis, “Molecular Geneticsof the Cyclic Nucleotide Phosphodiesterases”, pp. 227-241 in CyclicNucleotide Phosphodiesterases: Structure Regulation, and Drug Action,Beavo, J. and Houslay, M. D., Eds.; John Wiley & Sons, New York; 1990.See also, e.g., Faure et al., PNAS (USA), 85:8076 (1988)—D. discoideum;Sass et al., PNAS (USA), 83:9303 (1986)—S. cerevisiae, PDE class IV,designated PDE2; Nikawa et al., Mol. Cell. Biol., 7:3629 (1987)—S.cerevisiae, designated PDE1; Wilson et al., Mol. Cell. Biol., 8:505(1988)—S. cerevisiae, designated SRA5; Chen et al., PNAS (USA), 83:9313(1986)—D. melanogaster, designated dnc⁺; Ovchinnikow et al., FEBS,22:169 (1987)—bovine retina, designated GMP PDE; Davis et al., PNAS(USA), 86:3604 (1989)—rat liver, designated rat dnc-1; Colicelli et al.,PNAS (USA), 86:3599 (1989)—rat brain, designated DPD; Swinnen et al.,PNAS (USA), 86:5325 (1989)—rat testis, rat PDE1, PDE2, PDE3 and PDE4;and Livi et al., Mol. Cell. Biol., 10:2678 (1990)—human monocyte,designated hPDE1. See also, LeTrong et al., supra and Thompson et al.,supra.

Complementation screening has been used to detect and isolate mammaliancDNA clones encoding certain types of PDEs. Colicelli et al., PNAS(USA), 86:3599 (1989), reported the construction of a rat brain cDNAlibrary in an S. cerevisiae expression vector and the isolationtherefrom of genes having the capacity to function in yeast to suppressthe phenotypic effects of RAS2^(vall9), a mutant form of the RAS2 geneanalogous to an oncogenic mutant of the human HRAS gene. A cDNA socloned and designated DPD (rat dunce-like phosphodiesterase) has thecapacity to complement or “rescue” the loss of growth control associatedwith an activated RAS2^(vall9) gene harbored in yeast strain TK161-R2V(A.T.C.C. 74050), as well as the analogous defective growth controlphenotype of the yeast mutant 10DAB (A.T.C.C. 74049) which is defectiveat both yeast PDE gene loci (pde⁻¹, pde⁻²). The gene encodes ahigh-affinity cAMP specific phosphodiesterase, the amino acid sequenceof which is highly homologous to the cAMP-specific phosphodiesteraseencoded by the dunce locus of Drosophila melanogaster.

Through the date of filing of parent application Ser. No. 07/688,356,there have been no reports of the cloning and expression of DNAsequences encoding any of the mammalian Ca²⁺/calmodulin stimulated orcGMP-stimulated PDEs (PDE families I and II) and, accordingly, therecontinues to exist a need in the art for complete nucleotide sequenceinformation for these PDEs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel purified and isolatedpolynucleotide sequences (e.g. DNA and RNA including sense and antisensestrands) which code for expression of mammalian species (e.g., human andbovine) Ca²⁺/calmodulin stimulated cyclic nucleotide phosphodiesteraseand cGMP stimulated cyclic nucleotide phosphodiesterase polypeptides.Genomic and cDNA sequences provided by the invention may be associatedwith homologous or heterologous species expression control DNA sequencessuch as promoters, operators, regulators, terminators and the like toallow for in vivo and in vitro transcription to messenger RNA and, inturn, translation of mRNAs to provide functional phosphodiesterases andrelated polypeptides in large quantities.

Specifically provided by the invention are mammalian DNA sequencesencoding phosphodiesterases and fragments thereof which are present asmammalian DNA inserts in bacterial plasmids and viral vectors which arethe subject of deposits made with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852 on Apr. 11 and 15, 1991 andon Apr. 14, 1992 in accordance with U.S. Patent and Trademark Office andBudapest Treaty requirements. DNAs deposited in connection with thepresent invention include:

1. Plasmid pCAM-40 in E. coli (A.T.C.C. accession No. 68576) containinga bovine brain cDNA insert encoding a 61 kDa CaM-PDE isozyme;

2. Plasmid p12.3A in E. coli (A.T.C.C. 68577) containing a bovine braincDNA insert encoding a 63 kDa CaM-PDE isozyme;

3. Bacteriophage λ CaM H6a (A.T.C.C. accession No. 75000) containing ahuman hippocampus cDNA insert fractionally encoding a 61 kDa CaM-PDEisozyme;

4. Plasmid pHcam61-6N-7 in E. coli (A.T.C.C. accession No. 68963)containing a composite human cDNA insert encoding a 61 kDa CaM-PDEisozyme;

5. Plasmid pcamH3EF in E. coli (A.T.C.C. accession No. 68964) containinga human hippocampus cDNA insert encoding a novel PDE homologous to a 61kDa CaM-PDE;

6. Plasmid pcamHella in E. coli (A.T.C.C. accession No. 68965)containing a human heart cDNA insert encoding a novel PDE homologous toa 61 kDa CaM-PDE;

7. Plasmid p3CGS-5 in E. coli (A.T.C.C. accession No. 68579) containinga bovine adrenal cDNA insert encoding a cGS-PDE isozyme;

8. Plasmid pBBCGSPDE-5 in E. coli (A.T.C.C. accession No. 68578)containing a bovine brain cDNA insert encoding a cGS-PDE isozymefragment;

9. Plasmid pBBCGSPDE-7 in E. coli (A.T.C.C. accession No. 68580)containing a bovine brain cONA encoding a cGS-PDE isozyme;

10. Plasmid pGSPDE6.1 in E. coli (A.T.C.C. accession No. 68583)containing a human heart cDNA encoding a cGS-PDE isozyme fragment;

11. Plasmid pGSPDE7.1 in E. coli (A.T.C.C. accession No. 68585)containing a human hippocampus cDNA insert encoding a cGS-PDE isozymefragment; and

12. Plasmid pGSPDE9.2 (A.T.C.C. accession No. 68584) containing a humanhippocampus cONA insert encoding a cGS-PDE isozyme fragment. 13. PlasmidpHcgs6n in E. coli (A.T.C.C. accession No. 68962) containing a humancDNA insert encoding a cGS-PDE.

Also specifically provided by the present invention is a bovine cDNAsequence containing nucleotides encoding bovine 59 kDa CaM-PDE andcharacterized by the DNA and amino acid sequences of SEQ ID NO: 16 andSEQ ID NO: 17.

In related embodiments, the invention concerns DNA constructs whichcomprise a transcriptional promoter, a DNA sequence which encodes thePDE or a fragment thereof, and a transcriptional terminator, eachoperably linked for expression of the enzyme or enzyme fragment. Theconstructs are preferably used to transform or transfect host cells,preferably eukaryotic cells, and more preferably mammalian or yeastcells. For large scale production, the expressed PDE can be isolatedfrom the cells by, for example, immunoaffinity purification.

Incorporation of DNA sequences into procaryotic and eucaryotic hostcells by standard transformation and transfection processes, potentiallyinvolving suitable DNA and RNA viral vectors and circular DNA plasmidvectors, is also within the contemplation of the invention and isexpected to provide useful proteins in quantities heretofore unavailablefrom natural sources. Systems provided by the invention includetransformed E. coli cells, including those referred to above, as well asother transformed eukaryotic cells, including yeast and mammalian cells.Use of mammalian host cells is expected to provide for suchpost-translational modifications (e.g., truncation, lipidation, andtyrosine, serine or threonine phosphorylation) as may be needed toconfer optimal biological activity on recombinant expression products ofthe invention.

Novel protein products of the invention include expression products ofthe aforementioned nucleic acid sequences and polypeptides having theprimary structural conformation (i.e., amino acid sequence) of CaM-PDEand cGS-PDE proteins, as well as peptide fragments thereof and syntheticpeptides assembled to be duplicative of amino acid sequences thereof.Proteins, protein fragments, and synthetic peptides of the invention areprojected to have numerous uses including therapeutic, diagnostic, andprognostic uses and will provide the basis for preparation of monoclonaland polyclonal antibodies specifically immunoreactive with the proteinsof the invention.

Also provided by the present invention are antibody substances(including polyclonal and monoclonal antibodies, chimeric antibodies,single chain antibodies and the like) characterized by their ability tobind with high immunospecificity to the proteins of the invention and totheir fragments and peptides, recognizing unique epitopes which are notcommon to other proteins. The monoclonal antibodies of the invention canbe used for affinity purification of CaM-PDEs and cGS-PDEs, e.g., Hansenet al., Meth. Enzymol., 15:543 (1988).

Also provided by the present invention are novel procedures for thedetection and/or quantification of normal, abnormal, or mutated forms ofCaM-PDEs and cGS-PDEs, as well as nucleic acids (e.g., DNA and mRNA)associated therewith. Illustratively, antibodies of the invention may beemployed in known immunological procedures for quantitative detection ofthese proteins in fluid and tissue samples, and of DNA sequences of theinvention that may be suitably labelled and employed for quantitativedetection of mRNA encoding these proteins.

Among the multiple aspects of the present invention, therefore, is theprovision of (a) novel CaM-PDE and cGS-PDE encoding polynucleotidesequences, (b) polynucleotide sequences encoding polypeptides having theactivity of a mammalian CaM-PDE or of a mammalian cGS-PDE whichhybridize to the novel CaM-PDE and cGS-PDE encoding sequences underhybridization conditions of the stringency equal to or greater than theconditions described herein and employed in the initial isolation ofcDNAs of the invention, and (c) polynucleotide sequences encoding thesame (or allelic variant or analog polypeptides) through use of, atleast in part, degenerate codons. Correspondingly provided are viral DNAand RNA vectors or circular plasmid DNA vectors incorporatingpolynucleotide sequences and procaryotic and eucaryotic host cellstransformed or transfected with such polynucleotide sequences andvectors, as well as novel methods for the recombinant production ofthese proteins through cultured growth of such hosts and isolation ofthe expressed proteins from the hosts or their culture media.

In yet other embodiments, the invention provides compositions andmethods for identifying compounds which can modulate PDE activity. Suchmethods comprise incubating a compound to be evaluated for PDEmodulating activity with eukaryotic cells which express a recombinantPDE polypeptide and determining therefrom the effect of the compound onthe phosphodiesterase activity provided by gene expression. The methodis effective with either whole cells or cell lysate preparations. In apreferred embodiments the eukaryotic cell is a yeast cell or mammaliancell which lacks endogenous phosphodiesterase activity. The effect ofthe compound on phosphodiesterase activity can be determined by means ofbiochemical assays which monitor the hydrolysis of cAMP and/or cGMP, orby following the effect of the compound on the alteration of aphenotypic trait of the eukaryotic cell associated with the presence orabsence of the recombinant PDE polypeptide.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description thereof whichincludes numerous illustrative examples of the practice of theinvention, reference being made to the drawing wherein:

FIG. 1 provides the results of amino acid sequence determinations forisolated 59 kDa (bovine heart) and 63 kDa (bovine brain) CaM-PDEproteins in alignment with the complete sequence of the 61 kDa (bovinebrain) isozyme. Identities of the 59 and 63 kDa proteins to the 61 kDaisozyme are underlined. Tentative identifications are in lower cases andhyphens denote unidentified residues. The N-terminus of the 59 kDaisozyme, as determined by the subtraction of a methionyl peptide(mDDHVTIRRK) from the composition of an amino-terminal blocked lysylpeptide, is in parenthesis. Solid boxes are placed above residues withinthe CaM-binding sites identified in the 61 and 59 kDa isozymes.

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate practice of the invention. Example Irelates to the isolation, purification, and sequence determination of 61kDa CaM-PDE cDNA from bovine brain and to the expression thereof in amammalian host cell. Example II relates to the isolation, purification,and sequence determination of a 59 kDa CaM-PDE from bovine lung and tothe expression thereof in a mammalian host cell. Example III relates tothe isolation, purification, and sequence determination of 63 kDaCaM-PDE cDNA from bovine brain and to the expression thereof in amammalian host cell. Example IV relates to the isolation, purification,and sequence determination of cGS-PDE cDNA from bovine adrenal cortex,as well as the expression of the DNA in mammalian host cells. Example Vrelates to the isolation, purification, and sequence determination ofcGS-PDE cDNA from bovine brain and to the expression thereof in amammalian host cell. Example VI relates to the use of cGS-PDE bovineadrenal cDNA to obtain human cGS-PDE cDNAs and to the development of ahuman cDNA encoding a cGS-PDE. Example VII relates to the use of CaM-PDE61 kDa bovine brain cDNA to obtain a human CaM-PDE 61 kDa cDNA and anovel structurally related cDNA. Example VIII relates to the expressionof bovine and human PDE cDNAs for complementation of yeast phenotypicdefects and verification of phosphodiesterase activity for theexpression product. Example IX relates to tissue expression studiesinvolving Northern analysis and RNase protection studies employingpolynucleotides (specifically cDNAs and antisense RNAs) of theinvention.

In those portions of the text addressing the formation of redundantoligonucleotides, the following Table I single letter coderecommendations for ambiguous nucleotide sequence, as reported in J.Biol. Chem. 261:13-17 (1986), are employed: TABLE I Symbol MeaningOrigin of designation G G Guanine A A Adenine T T Thymine C C Cytosine RG or A puRine Y T or C pYrimidine M A or C aMino K G or T Keto S G or CStrong interaction (3 H bonds) W A or T Weak interaction (2 H bonds) HA, C, or T not G, as H follows G in the alphabet B G, C, or T not A V A,C, or G not T, (not U) as V follows U D A, G, or T not C N A, C, G, or Tany Nucleotide base

EXAMPLE I Isolation, Purification, and Sequence Determination of 61 kDaCaM-PDE cDNA From Bovine Brain

In this Example, a cDNA sequence representing that portion of a gene for61 kDa bovine brain CaM-PDE which encodes the amino terminus of theprotein was isolated by PCR from a collection of first strand cDNAsdeveloped from bovine brain mRNA. The PCR-generated fragment was thenemployed to isolate a full length bovine brain CaM-PDE sequence.

Total RNA was prepared from bovine heart using the method of Chomczynskiet al., Anal. Biochem., 162:156-159 (1987) and mRNA was selected using aPoly(A) Quik™ mRNA purification kit according to the manufacturer'sprotocol. First strand cDNA was synthesized by adding 80 units of AMVreverse transcriptase to a reaction mixture (40 μl, final volume)containing 50 mM Tris HCl (pH8.3 @ 42°), 10 mM MgCl₂, 10 mMdithiothreitol, 0.5 mM (each) deoxynucleotide triphosphates, 50 mM KCl,2.5 mM sodium pyrophosphate, 5 μg deoxythymidylic acid oligomers (12-18bases) and 5 μg bovine heart mRNA denatured for 15 min at 65°.Incorporation of 1 μl [³²P]-labeled dCTP (3000 Ci/mmol) was used toquantitate first strand cDNA synthesis. The reaction was incubated at42° for 60 min. The reaction was phenol/CHCl₃ extracted and EtOHprecipitated. The nucleic acid pellet was resuspended in 50 μl of 10 mMTris-HCl (pH 7.5)/0.1 mM EDTA to a final concentration of 15 ng per μl.

Redundant sense and antisense oligomers corresponding to 61 kDa peptidesequences as in FIG. 1 were designed to be minimally redundant, yet longenough to specifically hybridize to the target template.

A first 23 base oligomer, designated CaM PCR-2S, was synthesized on anApplied Biosystems, Inc. DNA synthesizer. The oligomer had the followingsequence, 5′-AARATGGGNATGAARAARAA-3′ SEQ ID NO:1

which specifies the following amino acid sequence, KMGMMKKK. SEQ ID NO:2

A second 23 base oligomer, designated CaM PCR-3AS, was synthesized withthe following sequence, 5′-ACRTTCATYTCYTCYTCYTGCAT-3′ SEQ ID NO:3

representing the following amino acid sequence, MQEEEMNV. SEQ ID NO:4

A 612 bp CaM PDE cDNA fragment was synthesized using the PCRamplification technique by adding 15 ng of first strand cDNA to areaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.5 mMMgCl₂, 0.01% gelatin, 0.1% Triton X-100, 0.2 mM (each) deoxynucleotidetriphosphates, 1 μM (each) CaM PCR 2S and CaM PCR-3AS oligomers, and 2.5units of Thermus aquaticus DNA polymerase. The reaction was incubatedfor 30 cycles as follows: 94° for 1 min; 50° for 2 min; and 72° for 2min. The reaction products were purified on a 1% agarose gel using 0.04M Tris-acetate/0.001 M EDTA buffer containing 0.5 μg/ml ethidiumbromide. The DNA products were visualized with UV light, cleanly excisedfrom the gel with a razor blade, purified using Geneclean II reagent kitand ligated into Eco RV-cut pBluescript vector DNA.

To determine if the PCR amplification products were CaM PDE cDNAs, thesubcloned PCR DNA products were sequenced from the ends using T3 and T7promoter primers and either Sequenase or Taq Polymerase sequencing kits.Approximately 250 bases from each end of this piece of DNA weresequenced and the deduced amino acid sequence from the cDNA correspondedwith the FIG. 1 amino acid sequences of the 59 and 61 kDa CaM-PDEs,confirming that the PCR DNA product was a partial CaM PDE cDNA.

A bovine brain cDNA library constructed with the lambda ZAP vector(kindly provided by Ronald E. Diehl, Merck, Sharp & Dohme) was screenedwith the radiolabeled 615 bp CaM-PDE cDNA obtained by PCR amplification.The probe was prepared using the method of Feinberg et al., Anal.Biochem., 137:266-267 (1984), and the [³²P]-labeled DNA was purifiedusing Elutip-D® columns. Plaques (700,000 plaques on 12-150 mm plates)bound to filter circles were hybridized at 42° C. overnight in asolution containing 50% formamide, 20 mM Tris-HCl (pH 7.5), 1×Denhardt's solution, 10% dextran sulfate, 0.1% SDS and 10⁶ cpm/ml[³²P]-labeled probe (10⁹ cpm/μg). The filters were washed three timesfor 15 min with 2×SSC/0.1% SDS at room temperature, followed by two15-min washes with 0.1×SSC/0.1% SDS at 45° C. The filters were exposedto x-ray film overnight.

Of the fifty-six plaques that hybridized with the [³²P]-labeled probeseight randomly selected clones were purified by several rounds ofre-plating and screening [Maniatis et al., Molecular Cloning: ALaboratory Manual 545 pp. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., (1982)] and the insert cDNA's were subcloned intopBluescript SK(−) by in vivo excision [Short et al., Nuc. Acids Res.,16:7583-7599 (1988)] as recommended by the manufacturer.

Plasmid DNA prepared from cultures of each clone were subjected torestriction analysis using EcoRI. Two clones of a suitable length wereselected for sequence analysis using Taq Tak® and Sequenase® sequencingkits. The two clones were pCaM-40 (2.3 kb) and pCaM-34 (2.7 kb). Thesequencing information from this procedure confirmed that the insert ofpCaM-40 encoded the full length bovine brain 61 kDa CaM-PDE. Thesequence of this clone and the amino acid sequence deduced therefrom areset forth in SEQ ID NO: 5 and SEQ ID NO: 6.

Transient expression of the 61 kDa CaM-PDE cDNA in COS-7 cells (A.T.C.C.CRL 1651) was accomplished as follows. Vector pCDM8 [Seed, Nature,32:840-843 (1987)] in E. coli host cells MC1061-p3 was generouslyprovided by Dr. Brian Seed, Massachusetts General Hospital, Boston,Mass. This vector is also available from Invitrogen, Inc. (San Diego,Calif.). Plasmid pCaM-40 was digested with HindIII and NotI, yielding a2.3 kb fragment which was ligated into CDM8 vector DNA which had beendigested with HindIII and NotI. The resulting plasmid was propagated inMC1061-p3 cells. Plasmid DNA was prepared using the alkaline lysismethod of Ausubel et al., eds., Current Protocols in Molecular Biology,1:1.7.1 (John Wiley & Sons, New York, 1989) and purified usingQiagen-Tip 500 columns (Qiagen, Inc. Chatsworth, Calif.) according tothe manufacturer protocol.

COS-7 cells were transfected with the p-CaM-40/CDM8 construct (or mocktransfected with the CDM8 vector alone) using the DEAE-dextran methodAusubel et al., supra at 1:9.2 et seq. Specifically, 10 μg of ethanolprecipitated DNA was resuspended in 80 μl TBS buffer, and added to 160μl of 10 mg per ml DEAE-dextran dropwise to a 100 mm plate of 50%confluent COS-7 cells in 4 ml of DMEM supplemented with 10% NuSerum, andmixed by swirling. The cells were incubated for 3-4 hours at 37° in awater-saturated 7% CO₂ atmosphere. The medium was removed and the cellswere immediately treated with 10% DMSO in PBS for 1 minute. Followingthis treatment, the cells were washed with PBS, then DMEM, and finallycultured in DMEM supplemented with 10% fetal bovine serum andantibiotics (50 μg/ml streptomycin sulfate) in a 7%-CO₂ incubator for 36hours.

COS cells were scraped from the plates and homogenized in a buffercontaining 40 mM Tris-HCl (pH=7.5), 5 mM EDTA, 15 mM benzamidine, 15 mMbeta-mercaptoethanol, 1 μg per ml pepstatin A and 1 μg per ml peupeptinusing a Dounce homogenizer (1 ml per 100 mm plate). Homogenates wereassayed for PDE activity according to the procedures of Hanson et al.,Proc. Nat'l. Acad. Sci., U.S.A., 79:2788-2792 (1982), using [³H]cGMP asthe substrate. Reactions were carried out at 30° for 10 minutes in abuffer containing 20 mM Tris-HCl (pH=7.5), 20 mM imidazole (pH=7.5), 3mM MgCl₂, 15 mM Mg acetate, 0.2 mg per ml BSA and 1 μM ³H-cAMP witheither 2 mM EGTA or 0.2 mM CaCl₂ and 4 Mg per ml CaM. Assays werestopped by incubating the tubes in a 900 water bath for 1 minute. Aftercooling, 10 μl of 2.5 mg per ml snake venom was added to each assay andincubated at 37° for 5 minutes. The samples were diluted with 250 μl of20 mM Tris-HCl (pH=7.5) and immediately applied to 0.7 ml A-25 ionexchange columns. The columns were washed three times with 0.5 ml of 20mM Tris-HCl (pH=7.5) and the eluate was collected in scintillationvials. Samples were counted for 1 minute using a Packard Model 1600TRscintillation counter. Specific cyclic nucleotide hydrolytic activitywas expressed as picomoles cAMP or cGMP hydrolyzed per minute per mgprotein. Protein concentration was estimated according to the method ofBradford, Anal. Biochem., 72:248-254 (1976), using BSA as a standard.When compared to mock transfected cells, extracts of cells transfectedwith pCaM-40 cDNA contained significantly greater CaMP and cGMPhydrolytic activities in the presence of EGTA. Assays of the pCaM-40cDNA-transfected cells in the presence of calcium and CaM resulted instimulation of cAMP and cGMP hydrolysis.

EXAMPLE II Isolation, Purification, and Sequence Determination of a 59kDa CaM-PDE From Bovine Lung

A fully degenerate sense oligonucleotide corresponding to the amino acidsequence MDDHVTI SEQ ID NO:7

from the bovine heart 59 kDa CaM-pde was synthesized. The nucleotidesequence of this oligonucleotide is 5′-ATGAGRAGRCAYGTHACGAT-3′. SEQ IDNO:8

An antisense oligonucleotide was designed from the FIG. 1 sequence ofbovine brain 61 kDa CaM-PDE, corresponding to the amino acid sequenceLRCLVKQ SEQ ID NO:9

and having the sequence, 5′-CTGCTTCACTAAGCATCTTAG-3′. SEQ ID NO:10

This primer pair was used to prime a PCR reaction using bovine heartfirst strand cDNA (as prepared in Example I) as a template. Thispredicted a PCR product of 75 bp, 54 bp of which were unique 59 kDasequence and 21 bp of which were shared between the 59 kDa and 61 kDaisozymes. The PCR products were analyzed by sieving agarose gelelectrophoresis, and a band migrating at 75 bp was excised from the gel.The DNA was subcloned into pBluescript KS⁺, and colonies positive by theblue/white selection scheme were screened by PCR using primers directedagainst vector sequences. Colonies with inserts of the appropriate sizewere selected, and one of these (pCaM59/75.14) was chosen forsequencing. Plasmid DNA was prepared using a Qiagen P20 push column andused as template for sequencing using the dideoxy method. The sequenceof the PCR product is SEQ ID NO:115′-ATGAGAAGGCACGTAACGATCAGGAGGAAACATCTCCAAAGACCCATCTTT-AGACTAAGATGCTTAGTGAAGCAG-3′.

Analysis of the sequence revealed differences in two codons between thesequence obtained and the predicted sequence. Re-examination of thesense oligonucleotide primer sequence revealed that an inadvertenttransposition of two codons had led to a mistake in the design of theoligonucleotide. A second set of oligonucleotide PCR primers wasprepared which predicted a 54 bp product with minimum overlap betweenthe 59 and 61 kDa isozymes; in addition, the second sense primerincorporated a correction of the mistake in the design of the originalsense primer. The sense oligonucleotide had the sequence5′-ATGGAYGAYCACGTAACGATC-3′ SEQ ID NO:12

and the antisense oligonucleotide had the sequence5′-AAGTATCTCATTGGAGAACAG-3′ SEQ ID NO:13

This primer pair was used to prime a PCR reaction using bovine heartfirst-strand cDNA as template and the PCR products subcloned andscreened exactly as described above. Two clones (pCaM59/54.9 andpCaM59/54.10) were selected for sequencing based on insert size andsequenced as described above; both clones contained 54 bp inserts of thepredicted sequence SEQ ID NO:145′-ATGGATGATCACGTAACGATCAGGAGGAAACATCTCCAAAGACCCAT CT-TTAGA-3′,

predicting the amino acid sequence MDDHVTIRRKHLQRPIFR SEQ ID NO:15

A cDNA library was constructed from bovine lung mRNA and screened usingprocedures as described in Example IV, infra, with respect to screeningof a bovine adrenal cortex library. Approximately 1.2×10⁶ plaque-formingunits were probed with a ³²P-labelled, 1.6 kb EcoRI restrictionendonuclease-cleavage product of the pCaM-40 cDNA. This initialscreening produced 4 putative 59 kDA CaM-PDE cDNA clones. Preliminarysequence analysis indicated that one clone, designated p59KCAMPDE-2,contained the complete coding sequence of the putative 59 kDa CaM-PDE. Aseries of nested deletions were constructed from the p59KCAMPDE-2plasmid [See, Sonnenburg et al., J. Biol. Chem., 266 (26): 17655-17661(1991)], and the resultant templates were sequenced by an adaptation ofthe method of Sanger using the Taq DyeDeoxy™ Terminator Cycle SequencingKit and an Applied Biosystems Model 373A DNA Sequencing System. The DNAand deduced amino acid sequences are set out in SEQ. ID NO: 16 and 17,respectively. A large open reading frame within the cDNA encodes a 515residue polypeptide with an estimated molecular weight of ≈59kilodaltons that is nearly identical to the 61 kDa CaM-PDE amino acidsequence except for the amino-terminal 18 residues. Moreover, thepredicted amino acid sequence of the p59KCAMPDE-2 open reading frame isidentical to the available sequence of the 59 kDa CaM-PDE purified frombovine heart, Novack et al., Biochemistry, 30: 7940-7947 (1991). Theseresults indicate that the p59KCAMPDE-2 cDNA represents an mRNA speciesencoding the 59 kDa CaM-PDE.

Transient expression of the 59 kDa bovine lung PDE was accomplished asin Example I. Specifically, a 2.66 kb, EcoRI/blunt-ended fragment ofp59KCAMPDE-2 cDNA was subcloned into pCDM8 which had been digested withXhoI and blunt-ended. The recombinant plasmid, designatedp59KCAMPDE-2/CDM8, was used to transiently transfect COS-7 cells andextracts prepared from transfected COS-7 cells were assayed for CaM-PDEactivity using 2 μM cAMP. COS-7 cells transfected with the p59KCAMPDE-2cDNA yielded a cAMP hydrolytic activity that was stimulated 4-5 fold inthe presence of calcium and calmodulin. Mock transfected COS-7 cells hadno detectable calmodulin-stimulated cAMP hydrolytic activity.

EXAMPLE III Isolation, Purification, and Sequence Determination of 63kDa CaM-PDE cDNA From Bovine Brain

Multiple fully and partially redundant oligonucleotides corresponding tothe amino acid sequence reported in FIG. 1 were synthesized for use inattempting to obtain a cDNA clone for the 63 kDa CaM-PDE. Annealingtemperatures used for the polymerase chain reactions were varied between2 to 20° C. below the theoretical melting temperature for the lowestmelting oligonucleotide of each sense-antisense pair. Except for probes63-12s and 63-13a, which are discussed below, the PCR products of eachof the oligonucleotide pairs under a wide range of conditions gavemultiple ethidium bromide bands when agarose gel-electrophoresed. Use of63-12s and 63-13a resulted in a PCR product that coded for 63 kDaCaM-PDE when sequenced.

A fully redundant sense 23-mer oligonucleotide, designated 63-12s, wasassembled having the following sequence 5′ATHCAYGAYTAYGARCAYACNGG-3′ SEQID NO:18

based on an amino acid sequence, IHDYEHTG SEQ ID NO:19

which is conserved in the 61 kDa bovine CaM-PDEs (see FIG. 1). Apartially redundant antisense 32-mer oligonucleotide, designated 63-13a,had the sequence SEQ ID NO:20 5′-TCYTTRTCNCCYTGNCGRAARAAYTCYTCCAT-3′

and was based on the following conserved sequence in the 63 kDa CaM-PDE,MEEFFRQGDKE SEQ ID NO:21

Messenger RNA was prepared from bovine brain cerebral cortex and poly A⁺selected. First strand complementary DNA was produced using AMV or MMLVreverse transcriptase. De-tritylated oligonucleotides werephosphorylated using 1 mH [γ-³²P]ATP at 1×10⁶ cpm/nmol and T4polynucleotide kinase. After separation of 5′ ³²P-labelledoligonucleotides from free ATP using NENsorb 20 columns, each wassuspended as a 20 μM (5′ phosphate) stock and combined finally at 400 nMeach in the PCR. The reaction was run using 50 ng total cDNA and 200 μMdNTP to obtain about 1 μg of PCR product. The reaction had an initialdenaturation step at 94° C. for 5 min followed by 30 cycles of a 1 min94° C. denaturation, an annealing step at 50° C. for 1 min, and a 2 minextension step at 72° C. Under the reaction conditions, a singleethidium bromide-staining band of 450 base pairs was obtained on agarosegel electrophoresis of 100 ng of the PCR product. Five μg of 5′phosphorylated PCR product was ligated to 15 ng EcoRV-cut BluescriptKS(+) plasmid using T4 DNA ligase in 5% PEG-6000 for 12 h at 21° C.Putative positives of XL 1-blue transformations were white coloniesusing isopropyl thiogalactoside (IPTG) and bromo- chloro- indolylgalactoside (Xgal) for chromogenic selection. Such picks were sequencedusing T3 or T7 primers, dideoxynucleotide terminators, and Sequenase.

One resultant clone (p11.5B) had the nucleotide sequence and translatedamino acid sequence provided in SEQ ID NO: 22 and SEQ ID NO: 23,respectively. The codons for the amino acids YEH found inoligonucleotide 63-12s were replaced by codons for the amino acidsequence NTR in p11.5B. This was probably due to a contaminant in63-12s. Since the translated open reading frame (ORF) was similar tothat reported in FIG. 1 for the 63 kDa CaM PDE, p11.5B was used toscreen a bovine brain cDNA library for a full length cDNA clone.

A bovine brain cDNA library was constructed in λ ZAP II. First strandcDNA was treated with RNase H, E. coli DNA polymerase, and E. coli DNAligase to synthesize second strand cDNA. The cDNA was blunt-ended byT4-DNA polymerase; EcoRI sites in the cDNA were protected with EcoRImethylase and S-adenosyl methionine and EcoRI linkers were ligated onwith T4 DNA ligase. After EcoRI restriction endonuclease treatment, freelinkers were separated from the cDNA by gel filtration over SepharoseCL-4B. λ ZAP II arms were ligated onto the cDNA and packaged by an invitro Gigapack Gold packaging kit obtained from Stratagene. 9.5×10⁵recombinants were obtained with 5.8% nonrecombinant plaques as assessedby plating with IPTG and X-gal. The library was amplified once by theplate lysate method to obtain 1.4×10⁷ pfu/ml.

An initial screen of a total bovine brain cDNA library in λ ZAP II wasperformed. 700,000 pfu were screened using ³²P-labelled oligonucleotide63-1s at a hybridization and wash temperature of 40° C. Oligonucleotide63-1s was a fully redundant 23-mer having the sequence5′-AARAARAAYYTNGARTAYACNGC-3′ SEQ ID NO:24

corresponding to the amino acid sequence KKNLEYTA SEQ ID NO:25A total of 21 putative positives were picked. Subsequent rescreens wereimpeded by the very high background found using this screening method.Therefore, aliquots of each primary pick were pooled and 50,000 pfu ofthe pool were replated and rescreened with p11.5B radiolabelled byrandom primers and [α-³²P]dCTP. One positive was obtained,plaque-purified, and rescued as a plasmid p12.3a. Its DNA sequence isprovided in SEQ ID NO: 26. Subsequently, the bovine brain cerebralcortex library was screened further with p11.5B. Two further independentclones, p12.27.9 and p12.27.11, were obtained out of a primary screen of1.4×10⁶ pfu. They were plaque-purified and rescued for sequencing.

Clone p12.3a codes for a protein sequence with most of the alignedpeptides isolated from bovine 63 kDa CaM-PDE as shown in FIG. 1. SEQ IDNO: 26 and SEQ ID NO: 27 set forth the coding region (i.e., the 1844nucleotides of an approximately 2.5 kilobase insert) of p12.3a. Basenumbers 248-290 code for amino acid sequence QLENGEVNIEELKK, SEQ IDNO:28

while the comparable (FIG. 1) peptide has the sequence QLIPGRVNIISLKKSEQ ID NO:29

Base numbers 942-990 code for an amino acid sequence KSECAILYNDRSVLENSEQ ID NO:30

while the isolated (FIG. 1) peptide sequence is KDETAILYNDRTVLEN. SEQ IDNO:31None of the nonaligned 63 kDa peptide sequence is found in any readingframe of p12.3a; also, the molecular weight of the p12.3a open readingframe, as translated, is 60,951 not 63,000. Therefore, this cDNA mayrepresent an isozyme variant of the 63 kDa protein. The other twoindependent clones (p12.27.9 and p12.27.11) seem to have ORF sequenceidentical to p12.3a. The open reading frame of one clone begins atnucleotide number 823 of p12.3a and is identical to p12.3a through itstermination codon. The other clone starts at nucleotide 198 and isidentical to p12.3a throughout its length. None of the three clones hasthe anomalous NTR peptide sequence found in p11.5B; all three have YEHas the 61 kDa CaM PDE.

Transient expression of the 63kDa CaM-PDE cDNA in COS-7 cells wasaccomplished as follows. A fragment of the cDNA insert of plasmid p 12.3including the protein coding region of SEQ.ID NO: 26 and flanked byBamHI restriction sites was prepared by PCR. More specifically,oligonucleotides corresponding to base Nos. 94-117 (with the putativeinitiation codon) and the antisense of base Nos. 1719-1735 (withsequence immediately 3′ of the termination codon) of SEQ.ID NO. 26 weresynthesized with two tandem BamHI sites on their 5′ ends. The twoprimers had the following sequences: SEQ. ID NO:325′-GGATCCGGATCCCGCAGACGGAGGCTGAGCATGG-3′ SEQ.ID NO:335′-GGATCCGGATCCAGGACCTGGCCAGGCCCGGC-3′

The two oligonucleotides were used in a PCR cycling 30 times from a 1min incubation at 94° C. to a 2 min 72° C. incubation with a final 10min extension reaction at 72° C. The 100 μl reaction used 20 μM of eacholigonucleotide and 100 pg p12.3a as the template in order to produce 5μg 1665 base pair product.

The product was extracted once with an equal volume of 1:1phenol:chloroform, made 0.3 M with regard to sodium acetate, andprecipitated with two volumes of ethanol overnight. The precipitate wasdried, rehydrated into 50 μl, and the cDNA was digested with 5 unitsBamHI restriction endonuclease for one hour at 37° C. Afterwards, thesolution was extracted once with an equal volume of 1:1phenol:chloroform. The 1641 base pair cDNA with BamHI 5′ and 3′ ends waspurified from the aqueous layer using Qiagen Q-20 columns (Qiagen, Inc.,Chatsworth, Calif.) and the protocol given by the manufacturer.

The cut, purified PCR product was ligated into BamHI digested, alkalinephosphatase-treated Bluescript KS(+) plasmid. The ligation product wassubcloned into XL1 cells; resulting transformants were screened bysequencing. One transformant (designated p11.6.c6) was isolated with theBamHI insert oriented such that the Bluescript KS(+) HindIII restrictionsite was 30 bases 5′ to the sequence of the insert encoding theinitiation codon. This plasmid was digested with HindIII and XbaIrestriction endonucleases to release the 1689 base pair fragment. Thefragment was ligated into HindIII- and XbaI-digested CDM8 vector DNA asin Example I.

COS-7 cells were transfected with the p12.3.a/CDM8 construct or mocktransfected with the CDM8 vector alone using the DEAE-dextran method asdescribed in Example 1. A ratio of 10 μg DNA/400 μg DEAE-dextran wasused, with a final DEAE-dextran concentration in the media of 100 μg/ml.After 48 h, cells were suspended in 1 ml of homogenization buffer (40 mMTris HCl, pH=7.5, 15 mM benzamidine HCl, 15 mM 6-mercaptoethanol, 0.7μg/ml pepstatin A, 0.5 μg/ml leupeptin, and 5 mM Na₄EDTA) and disruptedon ice using a Dounce homogenizer. The homogenates were diluted ½ tomake a final 50% (v/v) glycerol for storage at −20° C. and used eitherto assay for phosphodiesterase activity or to determine proteinconcentration. CaM-dependent and independent activities were determinedas in Example 1. Cells transfected with a p12.3.a DNA had a 15-foldincrease in CaM-stimulated cAMP phosphodiesterase activity and a 12-foldincrease in CaM-stimulated cGMP phosphodiesterase activity over basallevels. Mock transfected COS-7 cells showed no PDE activity over basallevels even with CaM stimulation.

EXAMPLE IV Isolation, Purification, Sequence Determination, andExpression of cGS-PDE cDNA From Bovine Adrenal Cortex

Total RNA was prepared from bovine adrenal outer cortex using the methodof Chomczynski et al., supra. Polyadenylated RNA was purified from totalRNA preparations using the Poly(A) Quick™ mRNA purification kitaccording to the manufacturer's protocol. First strand cDNA wassynthesized by adding 80 units of AMV reverse transcriptase to areaction mixture (40 μl, final volume) containing 50 mM Tris-HCl (pH8.3@42°), 10 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM (each)deoxynucleotide triphosphates, 50 mM KCl, 2.5 mM sodium pyrophosphate, 5μg deoxythymidylic acid oligomers (12-18 bases) and 5 μg bovine adrenalcortex mRNA denatured for 15 min at 65° C. The reaction was incubated at42° C. for 60 min. The second strand was synthesized using the method ofWatson et al., DNA Cloning: A Practical Approach, 1:79-87 (1985) and theends of the cDNA were made blunt with T4 DNA polymerase. EcoRIrestriction endonuclease sites were methylated [Maniatis et al., supra]using a EcoRI methylase (Promega), and EcoRI linkers (50-fold molarexcess) were ligated to the cDNA using T4 DNA ligase. Excess linkerswere removed by digesting the cDNA with EcoRI restriction endonuclease,followed by Sepharose CL-4B chromatography. Ausubel et al., supra. ThecDNA (25-50 ng per μg vector) was ligated into EcoRI-digested,dephosphorylated ZAP® II (Stratagene) arms [Short et al., Nuc. AcidsRes., 16:7583-7599 (1988)] and packaged [Maniatis et al., supra] withGigapack® Gold extracts according to the manufacturer's protocol.

Initially, an unamplified bovine adrenal cortex cDNA library was madeand screened with a redundant 23-mer antisense oligonucleotide probesend-labeled with γ-[³²P]ATP and T4 polynucleotide kinase. The oligomerscorresponding to the amino acid sequences EMMMYHMK SEQ ID NO:34 andYHNWMHAF SEQ ID NO:35

were made using an Applied Biosystems model 380A DNA synthesizer. Theirsequences are as follows: 5′-TT CAT RTG RTA CAT CAT CAT YTC-3′ SEQ IDNO:36 5′-AA NGC RTG CAT CCA RTT RTG RTA-3′ SEQ ID NO:37

Duplicate nitrocellulose filter circles bearing plaques from 12confluent 150 mm plates (approximately 50,000 pfu/plate) were hybridizedat 45° C. overnight in a solution containing 6×SSC, 1× Denhardt'ssolution, 100 μg/ml yeast tRNA, 0.05% sodium pyrophosphate and 10⁶cpm/ml radiolabeled probe (>10⁶ cpm per pmol). The filters were washedthree times in 6×SSC at room temperature, followed by ahigher-stringency 6×SSC wash at 10° C. below the minimum meltingtemperature of the oligomer probes, and exposed to x-ray film overnight.

A single 2.1 kb cDNA clone (designated pcGS-3:2.1) was isolated andsequenced. The amino acid sequence enclosed by the large ORF of thisclone was identical to peptide sequences of the cGS-PDE purified fromthe supernatant fraction of a bovine heart homogenate. LeTrong et al.,supra.

A second, amplified, bovine adrenal cortex cDNA library was screenedusing the [³²P]-labeled CGS-3:2.1 partial cDNA, yielding a 4.2 kb cDNA(designated 3CGS-5).

The library was constructed, amplified as in Maniatis et al., supra,plated and screened with the bovine cDNA insert from clone CGS-3:2.1.The probe was prepared using the method of Feinberg et al., supra, andthe radiolabeled DNA was purified using Elutip-D® columns. Plaques(600,000 pfu on twelve 150 mm plates) bound to filter circles werehybridized at 42° C. overnight in a solution containing 50% formamide,20 mM Tris-HCl (pH 7.5, 1× Denhardt's solution, 10% dextran sulfate,0.11% SDS and 10⁶ cpm/ml [³²P]-labeled probe (10⁹ cpm/μg). The filterswere washed three times for 15 minutes with 2×SSC/0.1% SDS at roomtemperature, followed by two 15-minute washes with 0.1×SSC/0.1% SDS at45° C. The filters were exposed to x-ray film overnight. Ausubel et al.,supra.

From this initial screening, 52 putative clones were identified. Twentyof these clones were randomly selected, purified by several rounds ofre-plating and screening [Maniatis et al., supra] and the insert cDNAswere subcloned into pBluescript SK(−) by in vivo excision [Short et al.,supra] as recommended by the manufacturer. Plasmid DNA prepared fromthese clones were analyzed by restriction analysis and/or sequencing.From this survey, a 4.2 kb cDNA representing the largest open readingframe was identified. The cDNA inserts from the other putative cloneswere shorter, and appeared to be identical based on the nucleotidesequence of the insert ends.

Putative cGS-PDE cDNAs were sequenced by a modification of the Sangermethod [Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467] usingSequenase® or Taq Trak® kits as directed by the manufacturer. Templateswere prepared from the cDNAs by constructing a series of nesteddeletions [Henikoff, Gene, 28:351-359 (1984)] in the vector, pBluescriptSK(−) (Stratagene) using exonuclease III and mung bean nucleaseaccording to the manufacturer's protocol. In cases where overlappingtemplates were not attained by this method, the cDNAs were cleaved atconvenient restriction endonuclease sites and subcloned intopBluescript, or specific oligomers were manufactured to prime thetemplate for sequencing. Single-stranded DNA templates were rescued byisolating the DNA from phagemid secreted by helper phage-infected XL1cells harboring the pbluescript plasmid [Levinson et al., supra] asrecommended by the manufacturer (Stratagene). Homology searches ofGENBANK (Release 66.0), EMBL (Release 25.0), and NBRF nucleic acid(Release 36.0) and protein (Release 26.0) databases were conducted usingWordsearch, FASTA and TFASTA programs supplied with the GeneticsComputer Group software package Devereux et al., Nuc. Acids Res.,12:387-395 (1984).

The nucleotide sequence and deduced amino acid sequence encoded by thelarge open reading frame of p3CGS-5 cDNA clone insert is provided in SEQID NO: 38 and SEQ ID NO: 39. Starting with the first methionine codon,the cDNA encodes a 921 residue polypeptide with a calculated molecularweight of about 103,000. Although no stop codons precede this sequence,an initiator methionine consensus sequence [Kozak, J. Cell Biol.,108:229-241 (1989)] has been identified. The presence of 36 adenosineresidues at the 3′ end of the cDNA preceded by a transcriptiontermination consensus sequence [Birnstiel et al., Cell, 4:349-359(1985)] suggests that all of the 3′ untranslated sequence of the cGS-PDEmRNA is represented by this clone.

A putative phosphodiesterase-deficient (PPD) strain of S49 cells [Bourneet al., J. Cell. Physiol., 85:611-620 (1975)] was transientlytransfected with the cGS-PDE cDNA using the DEAE-dextran method. ThecGS-PDE cDNA was ligated into the unique BamHI cloning site in amammalian expression vector, designated pZEM 228, following azinc-inducible metallothionine promoter and prior to an SV40transcription termination sequence. The DNA was purified fromlarge-scale plasmid preparations using Qiagen pack-500 columns asdirected by the manufacturer. PPD-S49 cells were cultured in DMEMcontaining 10% heat-inactivated horse serum, 50 μg/ml penicillin G and50 μg/ml streptomycin sulfate at 37° C. in a water-saturated 7% CO₂atmosphere. Prior to transfections, confluent 100 mm dishes of cellswere replated at one-fifth of the original density and incubated for24-36 h. In a typical transfection experiment, PPD-S49 cells (50-80%confluent) were washed with Tris-buffered-saline and approximately 2×10⁷cells were transfected with 10 μg of DNA mixed with 400 μg ofDEAE-dextran in one ml of TBS. The cells were incubated at 37° C. for 1hr with gentle agitation every 20 min. Next, DMSO was added to a finalconcentration of 10% and rapidly mixed by pipetting up and down. After 2min, the cells were diluted with 15 volumes of TBS, collected bycentrifugation, and washed, consecutively with TBS and DMEM. The cellswere resuspended in complete medium and seeded into fresh 100 mm plates(1-2×107 cells/10 ml/plate). After 24 h, the cells were treated with TBSalone, or containing zinc sulfate (final concentration=125 μM) andincubated for an additional 24 h. The cells were harvested and washedonce with TBS. The final cell pellets were resuspended in two mls ofhomogenization buffer (40 mM Tris-HCl; pH 7.5, 15 mM benzamidine, 15 mMβ-mercaptoethanol, 0.7 μg/ml pepstatin A, 0.5 μg/ml leupeptin and 5 mMEDTA) and disrupted on ice using a dounce homogenizer. The homogenateswere centrifuged at 10,000×g for 5 min at 4° C. and the supernatantswere assayed for phosphodiesterase activity and protein concentration.

cGS PDE activity was determined by a previously described method using[³H]cAMP as the substrate as in Martins et al., J. Biol. Chem.,257:1973-1979 (1982). Phosphodiesterase assays were performed intriplicate. The Bradford assay [Bradford, Anal. Biochem., 72:248-254(1976)] was used to quantitate protein using BSA as the standard.

In the absence of zinc treatment, no increase in basal activity orcGMP-stimulated phosphodiesterase activity was detected in PPD S49 cellstransfected with the cGS PDE-ZEM 228 construct or the vector alone.However, zinc-treated cells transfected with cGS-PDE cDNA, but not thevector alone, expressed cGMP-enhanced cAMP phosphodiesterase activityindicating that the cDNA encodes a cGS-PDE. The total activity of thehomogenates and 50,000×g supernatants was not significantly different.

Transient expression of the CGS-PDE cDNA in COS-7 cells was accomplishedas in Example I. A 4.2 kb fragment of p3CGS-5 was isolated using HindIIIand NotI and was inserted into plasmid pCDM8, which had been digestedwith the same enzymes. The character of products produced in COS-7 cellstransformed with the p3CGS-5/pCDM8 construct is discussed in Example V,infra.

EXAMPLE V Isolation, Purification, and Partial Sequence Determination ofcGS-PDE cDNA from Bovine Brain

A. Isolation of Bovine Brain cGSPDE cDNA Clone. DBBCGSPDE-5

A bovine brain cDNA library constructed with the λ ZAP vector (kindlyprovided by Ronald E. Diehl, Merck, Sharp & Dohme) was screened with a450 bp EcoRI/ApaI restriction endonuclease cleavage fragment of thep3CGS-5 cDNA corresponding to (p3CGS-5) nucleotide position numbers1-452. The probe was prepared using the method of Feinberg et al.,supra, and the [³²P]-labeled DNA was purified using Elutip D® columns.Plaques (a total of 600,000 plaques on 12-150 mm plates) bound to filtercircles were hybridized at 42° overnight in a solution containing 50%formamide, 20 mM Tris HCl (pH 7.5), 1× Denhardt's solution, 10% dextransulfate, 0.1% SDS and 10⁶ cpm/ml [³²P]-labeled probe (10⁹ cpm/Ag). Thefilters were washed three times for 15 minutes with 2×SSC/0.1% at roomtemperature, followed by two 15 minute washes with 0.1×SSC/0.1% SDS at45%. The filters were exposed to x-ray film overnight.

Forty putative clones were picked from this first screen, of which sixwere randomly selected and purified by several rounds of re-plating andscreening [Maniatis et al., supra]. The insert cDNAs were subcloned intopBluescript SK(−) by in vivo excision as recommended by themanufacturer. Plasmid DNA prepared from cultures of each clone wassequenced from the ends using Sequenase and Taq Trak sequencing kits.The sequence obtained from this experiment confirmed that the bovinebrain cDNA clone, pBBCGSPDE-5 was a cGS-PDE cDNA, and that it wasdifferent than the adrenal cGS-PDE cDNA at the five-prime end.

Partial sequence analysis of the pBBCGSPDE-5 insert at its 5′ end(encoding the amino terminal region of the protein) revealed the sensestrand set out in SEQ ID NO: 40, while sequencing of the 3′ end of theinsert revealed the antisense sequence of SEQ ID NO: 41.

B. Isolation of Bovine Brain cGS-PDE cDNA Clone, pBBCGSPDE-7

Each of the forty putative clones selected from the first round ofpurification described above was spotted individually onto a lawn ofhost XL1 cells and incubated overnight at 37°. The plaques were screenedwith a 370 bp PstI/SmaI restriction endonuclease cleavage fragment ofthe p3CGS-5 cDNA (corresponding p3CGS-5 nucleotide position numbers2661-3034). The probe was prepared using the method of Feinberg et al.,supra, and the [³²P]-labeled DNA was purified using Elutip-D® columns.Plaques bound to filter circles were hybridized at 42° overnight in asolution containing 50% formamide, 20 mM Tris-HCl (pH 7.5), 1×Denhardt's solution, 10% dextran sulfate, 0.1% SDS and 10⁶ cpm/ml[³²P]-labeled probe (10⁹ cpm/μg). The filters were washed three timesfor 15 minutes with 2×SSC/0.1% SDS at room temperature, followed by two15-minute washes with 0.1×SSC/0.1% SDS at 45°. The filters were exposedto x-ray film overnight.

After several rounds of plating and rescreening, six putative cloneswere purified and sequenced from the ends. The sequence of thefive-prime end of the cDNA clone pBBCGSPDE-7 was identical to clonepBBCGSPDE-5, but not the adrenal gland-derived clone, p3CGS-5. Thesequence of the three-prime end of the pBBCGSPDE-7 cDNA clone wasidentical to the p3CGS-5 insert sequence.

Sequence analysis of the pBBCGSPDE-7 insert revealed the DNA sequenceset out in SEQ ID NO: 42 and the amino acid sequence of SEQ. ID NO: 43.

The large open reading frame encodes a 942-residue polypeptide that isnearly identical to the adrenal gland cGS-PDE isozyme (921 residues).The difference in the primary structure of these two isozymes lies inthe amino-terminal residues 1-46 of the brain cGS-PDE, and residues 1-25of the adrenal cGS-PDE. The remaining carboxy-terminal residues of thebrain and adrenal cGS-PDE are identical.

For transient expression in COS-7 cells, a 3.8 kb fragment ofpBBCGSPDE-7 was isolated using HindIII and NotI and inserted intoplasmid PCDM8 which had been cut with HindIII and NotI restrictionendonucleases. The recombinant pBBCGSPDE-7/CDM8 construct was used totransiently transfect COS-7 cells. The properties of thepBBCGSPDE-7/CDMS construct and the p3CGS-5/CDM8 construct prepared inExample IV products were subsequently compared. Membrane and supernatantfractions were prepared from extracts of transfected COS-7 cells andassayed for cGS-PDE activity. Both the pBBCGSPDE-7/CDM8 and p3CGS5/CDM8plasmid constructs produced cGS-PDE activities in COS-7 cell extracts,and most of the activity was detected in the supernatant fractions.However, a 10-fold greater percentage of total cGS-PDE activity wasdetected in membranes from COS-7 cell extracts transfected with thepBBCGSPDE-7/CDM8 construct than in membranes prepared fromp3CGS-5/CDM8-transfected COS-7 cells. These results indicate that,relative to the adrenal cGS-PDE, the isozyme encoded by the pBBCGSPDE-7cDNA preferentially associates with cellular membranes.

EXAMPLE VI Use of cGS-PDE Bovine Adrenal cDNA to Obtain Human cGS-PDEcDNAs

Several human cDNA clones, homologous to a cDNA clone encoding thebovine cyclic GMP-stimulated phosphodiesterase, were isolated byhybridization using a nucleic acid probe derived from the bovine cDNA. Acombination of sequence analysis and hybridization studies indicatesthat these human cDNA clones encompass an open reading framecorresponding to a human phosphodiesterase.

cDNA libraries were probed with DNA from plasmid p3cGS-5 which containsa 4.2-kb cDNA insert encoding the bovine cGS-PDE. This plasmid wasdigested with the restriction enzymes SmaI and EcoRI. The approximately3.0 kb fragment derived from the cDNA insert was isolated and purifiedby agarose gel electrophoresis. This fragment contains the entire openreadinq-frame of the PDE. The fragment was labeled with radioactivenucleotides by random priming.

The cDNA libraries were plated on a 150 mm petri dishes at a density ofapproximately 50,000 plaques per plate. Duplicate nitrocellulose filterreplicas were prepared. The radioactive nucleic acid probe was used forhybridization to the filters overnight at 42° C. in 50% formamide,5×SSPE (0.9 M NaCl, 0.05 M NaH₂PO₄H₂O, 0.04 M NaOH, and 0.005 MNa₂EDTA·₂H₂O), 0.5% SDS, 100 μg/ml salmon testes DNA, and 5× Denhardt'ssolution. The filters were washed initially at room temperature andsubsequently at 65° C. in 2×SSC containing 0.1% SDS. Positive plaqueswere purified and their inserts were subcloned into an appropriatesequencing vector for DNA sequence analysis by standard techniques.

First, a λgt10 cDNA library prepared from human hippocampus mRNA(clontech, random and dT primed) was screened. Of the approximately500,000 plaques examined, 33 hybridized to the probe. One of thesephages was digested with EcoRI to remove the cDNA insert. Thisinsert-containing EcoRI fragment was cloned into Bluescript KS that hadbeen digested. with EcoRI and then treated with calf intestine alkalinephosphatase. One product of this reaction was the plasmid pGSPDE9.2,which showed two major differences when compared to the bovine cGS-PDEcDNA. The 5′0.4 kb of the pGSPDE9.2 insert diverged from the bovinecDNA. Approximately 0.7 kb from the 5′ end of the human cDNA there is a0.7 kb region that diverges from the bovine cDNA. This region may be anintron. Twenty-five of the remaining hippocampus plaques that hadhybridized to the bovine probe were examined by PCR, hybridizationand/or sequencing. None were found to extend through the regions thatdiffered between the bovine and human cDNAs.

Phages λ GSPDE7.1 and λ GSPDE7.4, two other phages from the hippocampuslibrary, were digested with EcoRI and HindIII. Each yielded a 1.8-kbfragment that contains most of the cDNA insert and approximately 0.2-kbof phage lambda DNA. The λ DNA is present in the fragment because ineach case one of the EcoRI sites that typically bracket a cDNA inserthad been destroyed, possibly when the library was constructed. TheEcoRI/HindIII fragments were cloned into Bluescript KS digested withEcoRI and HindIII. This procedure gave rise to the plasmids pGSPDE7.1and pGSPDE7.4. The cDNA inserts encode DNA homologous to the 3′ portionof the bovine phosphodiesterase cDNA. Both of the cDNA inserts in theseclones begin at an EcoRI site and the sequences are homologous adjacentthis site.

Portions of pGSPDE7.1 and pGSPDE7.4 cDNA inserts were sequenced and areidentical except for a short region of their 3′ ends. The cDNA insert inpGSPDE7.1 ends with a sequence of approximately 70 adenine bases, whilethe cDNA insert in pGSPDE7.4 ends with three additional nucleotides notpresent in pGSPDE7.1 followed by a sequence of approximately 20 adeninebases.

Next, a cDNA library prepared in λ ZapII (Stratagene) from human heartmRNA yielded one hybridizing plaque from the approximately 500,000screened. The Bluescript SK(−) plasmid pGSPDE6.1 containing thehybridizing insert was excised in vivo from the λ ZapII clone. Sequenceanalysis showed that the insert is homologous to the bovinephosphodiesterase cDNA. The homologous region spans the position of theEcoRI found in the sequence formed by joining the sequence of the insertfrom pGSPDE9.2 to the sequence of the insert in pGSPDE7.1 or pGSPDE7.4.Thus, it is thought that the two clones from the hippocampus form acomplete open reading frame.

A third λ gt10 library derived from human placenta mRNA yielded fivehybridizing plaques from approximately 800,000 screened. These placentalcDNA clones were short and their sequences were identical to portions ofthe hippocampus cDNA pGSPDE9.2. Screening 5×10⁵ plaques from U118glioblastoma cDNA library, 5×10⁵ from a spleen cDNA library and 5×10⁵from an adrenal library (Cushings Disease) gave no hybridizationplaques.

Given the homology between the bulk of human and bovine cGS-PDEsequence, it was decided to obtain multiple independent cDNA clonescontaining the 5′ end of the human cGS-PDE to determine if the 0.4 kb 5′sequence was an artifact. An approximately 0.95 kb EcoRI-HindII fragmentfrom the 5′ end of the bovine cGS cDNA plasmid p3cgs5 was random primedand used as a probe to screen a number of human cDNA libraries.Hippocampus library screening was carried out under the same screeningconditions as described above. All remaining screenings were carried outas described with respect to human heart cDNA library screenings inExample VII, infra. No positives were obtained screening 5×10⁵ plaquesfrom a human T cell library (Hut78, dT-primed), 10⁶ plaques from thehippocampus cDNA library (random and dT-primed), 5×10⁵ plaques from ahuman liver cDNA library (dT-primed, 5′ stretch, Clontech), 5×10⁵plaques from a human SW1088 glioblastoma cDNA library (dT-primed), 5×10⁵plaques from the same heart cDNA library (random and dT-primed), and1.5×10⁶ plaques from a human lung cDNA library (random primed). Twopositives were obtained from screening 5×10⁵ plaques from a human fetalbrain cDNA library (random and dT-primed, Stratagene). These weredesignated as HFB9.1 and HFB9.2.

Bluescript SK(−) plasmids pHFB9.2 and pHFB9.1 were excised in vivo fromthe λZapII clones. DNA sequence analysis revealed that HFB9.1 startsabout 80 nucleotides further 3′ than does HFB9.2 and reads into anintron approximately 1.9 kb of the way into HFB9.2. HFB9.2 covers theentire open reading frame of the cGS-PDE, but reads into what may beganintron 59 nucleotides after the stop codon. Both of them lack the 5′0.4kb and the presumed intron found in pGSPDE9.2. The entire open readingframe of HFB9.2 was isolated and assembled into yeast expression vectorpBNY6N. The resulting plasmid, designated pHcgs6n, includes the codingregion of the cDNA as an EcoRI/XhoI insert. DNA and deduced amino acidsequences for the insert are provided in SEQ. ID No: 44 and 45,respectively.

EXAMPLE VII Use of CaM-PDE 61 kDa Bovine Brain cDNA to Obtain HumanCaM-PDE 61 kDa cDNA

Human cDNA clones, λ CaM H6a and λ CaM H3a, which are homologous to thecDNA encoding the bovine 61 kDa CaM-PDE, were obtained by hybridizationusing a nucleic acid probe derived from the cDNA encoding the bovinespecies enzyme. A combination of sequence analysis and hybridizationstudies indicate that λ Cam H6a contains most of an open reading frameencoding a human CaM-PDE.

The hybridization probe used to isolate the human DNA was derived fromfirst strand cDNA of bovine lung tissue by PCR treatment. Morespecifically, the 23-mer oligonucleotide designated PCR-2S in Example I(see, SEQ ID NO: 1) was combined in a PCR reaction with bovine lung cDNAand a redundant antisense 23-mer oligonucleotide (PCR-5AS) based on thepCaM insert sequence with 5′TCRTTNGTNGTNCCYTTCATRTT-3′ SEQ ID NO:46

representing the amino acid sequence NMKGTTND, SEQ ID NO:47according to the general procedures of Examples I and III, to generate a1098 bp cDNA fragment representing a large portion of the coding regionof the pCAM-40 insert. The PCR products were purified on a 1% agarosegel using 0.4 M Tris-acetate/0.001 M EDTA buffer containing 0.5 μg/mlethidium bromide. The DNA products were visualized with UV light,cleanly excised from the gel with a razor blade, purified usingGeneclean II reagent kit and ligated into EcoRV-cut pBluescript vectorDNA.

To determine if the PCR amplification products were CAM-PDE cDNAs, thesubcloned PCR DNA products were sequenced from the ends using T3 and T7promoter primers and either Sequenase or Taq Polymerase sequencing kits.Approximately 250 bases from each end of this DNA were then compared tothe amino acid sequence of bovine CAM-PDE, confirming that the PCR DNAproduct was a partial CAM PDE cDNA. This clone was designated pCAM-1000and contained a 1.1-kb insert of nucleic acid that corresponds tonucleotides 409 to 1505 of the insert of pCAM-40. pCaM1000 was digestedwith the restriction enzymes HinDIII and BamHI. The 1.1-kb fragment waspurified by agarose gel electrophoresis and then digested with therestriction enzyme AccI. The two fragments were separated and purifiedby agarose gel electrophoresis. These separated fragments were labeledwith radioactive nucleotides by random priming.

Human cDNA libraries were plated on 150 mm petri dishes at a density ofapproximately 50,000 plaques per dish and duplicate nitrocellulosefilter replicas were prepared. Each probe was hybridized to a separateset of the duplicate filters. The filters were hybridized overnight at65° C. in 3×SSC, 0.1% sarkosyl, 50 μg/ml salmon testes DNA, 10×Denhardt's solution, 20 mM sodium phosphate (pH 6.8). They were washedat 65° C. in 2×SSC containing 0.1% SDS.

A λ gt10 library prepared from human hippocampus mRNA yielded threehybridizing plaques of the approximately 500,000 screened. Of thesethree hybridizing plaques, two hybridized to both probes and the thirdhybridized to the longer of the two-probes. The λ Cam H6a clone containsan approximately 2 kb insert that is homologous to the cDNA encoding thebovine clone of pCAM-40.

The λ cam H6a cDNA was subcloned into the plasmid Bluescript KS forsequence analysis. Although the cDNA library had been constructed withEcoRI linkers, one of the EcoRI sites that should have flanked the cDNAinsert did not cut with EcoRI. Thus, the cDNA was subcloned as twofragments: an approximately 0.7 kb EcoRI/HindIII fragment (pcamH6C) andan approximately 1.6 kb HindIII fragment that contained approximately1.3 kb of cDNA and 0.25 kb of flanking λgt10 vector DNA (pcamH6B). DNAsequence analysis revealed that it encoded most of a human CaM-PDEhomologous to the bovine 61 k CaM-PDE, except that the human cDNAappeared to be missing two base pairs in the middle of the codingregion. These missing nucleotides correspond to positions 626 and 627 ofthe human cDNA sequence if it is aligned with the pCAM-40 bovine 61 kDaCaM-PDE (SEQ. ID NO: 5 for maximum homology.

Another of the cDNA clones from the hippocampus cDNA library that hadbeen screened with the bovine 61 kDa CaM-PDE probes was λcamH2a. Itcontained an approximately 1.0 kb insert. As was the case for λcamH6acDNA, only one of the two EcoRI sites that should be present at the endsof the insert would cut. The original subcloning and DNA sequenceanalysis for this cDNA utilized PCR fragments generated with oligos inthe flanking λgt10 vector arms. This cDNA overlaps much of the 5′ end ofthe insert in λcamH6a and contained the additional two nucleotidespredicted by the bovine sequence and required to maintain the PDE openreading frame. The λcamH2a insert also appeared to contain two introns;one 5′ of the initiator methionine and one downstream of the HindIIIsite. The EcoRI/HindIII fragment from λcamH2a (corresponding to theregion covered by pcamH6C) was subcloned into the plasmid Bluescript SK⁻and designated pcamH2A-16. This was then used as the source of the twoadditional bp in the construction of yeast expression plasmids describedbelow.

Two different plasmids were constructed for human CaM-PDE expression inyeast. One plasmid, pHcam61-6N-7, contains the entire open readingframe. The second plasmid, pHcam61met140, starts at an internalmethionine (beginning at nucleotide position 505) and extends to the endof the open reading frame. These expression plasmids were constructed bymodifying the 3′ portion of the open reading frame and then adding thetwo differently modified 5′ ends to the 3′ end. The sequence of the cDNAinsert of pHcam61-6N-7 is set out in SEQ. ID NO: 48 and the deducedamino acid sequence of the CaM-PDE encoded thereby is set out in SEQ. IDNO: 49. During construction of the cDNA insert, the nucleotide atposition 826 was altered from T to C, but the encoded amino acid wasconserved. Plasmid pHcam61met140, as noted above, has a cDNA insertlacking the first 140 codons of the coding region of the pHcam61-6N-7but is otherwise identical thereto.

A third cDNA, λcamH3a, contained an approximately 2.7 kb insert. ThiscDNA insert was subcloned for sequence analysis. Although the cDNAlibrary had been constructed with EcoRI, the inserted cDNA in λcamH3acould not be excised with EcoRI. Presumably one of the EcoRI sites wasdestroyed during the construction of the library. The cDNA insert wasexcised from the λ clone by digestion with HindIII and EcoRI. Thisdigestion yields two relevant fragments, a 0.6 kb HindIII fragment whichcontains a portion of DNA from the left arm of λgt10 attached to thecDNA insert and an approximately 2.4 kb HindIII/EcoRI fragmentcontaining the remainder of the cDNA insert. These two fragments wereassembled in the plasmid Bluescript KS to yield an approximately 3 kbfragment. The orientation of the small HindIII fragment was the same asthe original λ clone. This subclone is known as pcamH3EF. Although thiscDNA hybridizes to the bovine probe from the bovine CaM-PDE 61 kDa cDNA,sequence analysis revealed that it appeared to be the product of adifferent CaM-PDE gene. Plasmid pcamH3EF contains what may be the entireopen reading frame and would encode a protein approximately 75%homologous to the protein encoded by the insert of pHcam61-6N-7 overmuch of its lengths. DNA and deduced amino acid sequences are set out inSEQ. ID NOS: 50 and 51, respectively. The DNA sequence of the regionbetween nucleotide 80 and 100 of pcamH3EF is uncertain. This area is 5′to the initiator methionine codon and thus does not effect the openreading frame.

An approximately 2.4 kb fragment of pcamH3EF was gel purified followingdigestion with the restriction enzymes HindIII and EcoRI. This fragmentwas used to screen additional human cDNA libraries in a similar mannerto the screen described above. Screening approximately 5×10⁵ plaquesfrom a human heart cDNA library (Stratagene) yielded two plaques thathybridized to the pcamH3EF probe. The Bluescript SK⁻ plasmid pcamHellawas excised in vivo from one of these positive λZapII clones. DNA anddeduced amino acid sequences for the cDNA insert are set out in SEQ. IDNO: 52 and 53, respectively. Sequence analysis of pcamHella showed thatthe insert began at nucleotide position 610 of pcamH3EF and was nearlyidentical through nucleotide position 2066, at which point the DNAsequence diverged from that of pcamH3EF. The cDNA insert of pcamHellacontinued for approximately 0.6 kb. The consequence of this divergenceis to alter the carboxy terminus of the protein that would be encoded bythe open reading frame within the cONA. The pcamH3EF cDNA could encode aprotein of 634 amino acids (MW72,207). Assuming the 5′ end of thepcamHella cDNA is the same as that of the 5′ end of pcamH3EF (5′ tonucleotide position 610), pcamHella could encode a 709 amino acidprotein (MW80,759). These divergent 3′ ends may be the consequence ofalternative splicing, lack of splicing, or unrelated DNA sequences beingjuxtaposed during the cloning process.

EXAMPLE VIII Expression of Bovine and Human PDE cDNAs forComplementation of Yeast Phenotypic Defects

The present example relates to the expression of bovine and PDE clonesin yeast demonstrating the capacity of functional PDE expressionproducts to suppress the heat shock phenotype associated with mutationof yeast phosphodiesterase genes and also relates to the biochemicalassay of expression products. The host cells used in these procedureswere S. cerevisiae yeast strains 10DAB (ATCC accession No. 74049) andYKS45, both of which were pde¹⁻ pde²⁻ resulting in a phenotypecharacterized by heat shock sensitivity, i.e., the inability of cells tosurvive exposure to elevated temperatures on the order of 55-56° C. Inthese complementation procedures, the inserted gene product was noted toconspicuously modify the heat shock phenotype. This capacity, in turn,demonstrates the feasibility of. systems designed to assay chemicalcompounds for their ability to modify (and especially the ability toinhibit) the in vivo enzymatic activity of mammalian Ca²⁺/calmodulinstimulated and cGMP stimulated cyclic nucleotide phosphodiesterases.

A. Yeast Phenotype Complementation by Expression of a cDNA EncodingCaM-PDE

A 2.2 kb cDNA fragment, adapted for insertion into yeast expressionplasmids pADNS (ATCC accession No. 68588) and pADANS (ATCC accession No.68587) was derived from plasmid pCAM-40 (Example I) by polymerase chainreaction. Briefly, the following PCR amplification was employed to alterthe pCAM-40 DNA insert to align it appropriately with the ADH1 promoterin the vectors.

One oligonucleotide primer (Oligo A) used in the PCR reaction5′-TACGAAGCTTTGATGGGGTCTACTGCTAC-3′ SEQ ID NO:54

anneals to the pCaM-40 cDNA clone at base pair positions 100-116 andincludes a HindIII site before the initial methionine codon. A secondoligonucleotide primer (Oligo B) 5′-TACGAAGCTTTGATGGTTGGCTTGGCATATC-3′SEQ ID NO:55

was designed to anneal at positions 520-538 and also includes a HindIIIsite two bases before a methionine codon. The third oligonucleotide5′-ATTACCCCTCATAAAG-3′ SEQ ID NO:56annealed to a position in the plasmid that was 3′ of the insert. For onereaction, Oligo A and Oligo C were used as primers with pCAM-40 as thetemplate. The nucleic acid product of this reaction included the entireopen reading frame. A second reaction used Oligo B and Oligo C asprimers on the template pCaM-40 and yielded a nucleic acid product thatlacked the portion of the cDNA sequence encoding the calmodulin bindingdomain. These amplified products were digested with HindIII and NotI andligated to HindIII/NotI-digested yeast expression vectors pADNS andpADANS. Plasmid clones containing inserts were selected and transformedinto S. cerevisiae strain 10DAB by lithium acetate transformation.

Transformed yeast were streaked in patches on agar plates containingsynthetic medium lacking the amino acid leucine (SC-leucine agar) andgrown for 3 days at 30° C. Replicas of this agar plate were made withthree types of agar plates: one replica on SC-leucine agar, one replicaon room temperature YPD agar, and three replicas on YPD agar plates thathad been warmed to 56° C. The three warmed plates were maintained at 56°C. for 10, 20, or 30 minutes. These replicas were than allowed to coolto room temperature and then all of the plates were placed at 30° C.Yeast transformed with plasmids constructed to express the CaM-PDE wereresistant to the thermal pulse. More specifically, both the constructdesigned to express the complete open reading frame and that designed toexpress the truncated protein (including the catalytic region but notthe calmodulin binding domain), in either pADNS or pADANS, complementedthe heat shock sensitivity phenotype of the 10DAB host cells, i.e.,rendered them resistant to the 56° C. temperature pulse.

In a like manner, plasmids pHcam61-6N-7 and pHcam61met140 (Example VII)were transformed into yeast host 10DAB. Heat shock phenotypes weresuppressed in both transformants.

B. Biochemical Assay of Expression Products

The bovine CaM-PDE expression product was also evaluated by preparingcell-free extracts from the 10DAB yeast cells and measuring theextracts' biochemical phosphodiesterase activity. For this purpose, 200ml cultures of transformed yeast were grown in liquid SC-leucine to adensity of about 6 million cells per ml. The cells were collected bycentrifugation and the cell pellets were frozen. Extracts were preparedby thawing the frozen cells on ice, mixing the cells with 1 ml of PBSand an equal volume of glass beads, vortexing them to disrupt the yeastcells, and centrifuging the disrupted cells at approximately 12,000×gfor 5 min to remove insoluble debris. The supernatant was assayed forphosphodiesterase activity.

Extracts of yeast cells, up to 50 μl, were assayed for phosphodiesteraseactivity in 50 mM Tris (pH 8.0), 1.0 mM EGTA, 0.01 mg/mL BSA (bovineserum albumin), [³H]-cyclic nucleotide (4-10,000 cpm/pmol), and 5 mMMgCl₂ in a final volume of 250 μl at 30° C. in 10×75 mm glass testtubes. The incubations were terminated by adding 250 μl of 0.5 M sodiumcarbonate pH 9.3, 1M NaCl, and 0.1% SDS. The products of thephosphodiesterase reaction were separated from the cyclic nucleotide bychromatography on 8×33 mm columns of BioRad Affi-Gel 601 boronic acidgel. The columns were equilibrated with 0.25M sodium bicarbonate (pH9.3) and 0.5 M NaCl. The reactions were applied to the columns. Theassay tubes were rinsed with 0.25M sodium bicarbonate (pH 9.3) and 0.5 MNaCl and this rinse was applied to the columns. The boronate columnswere washed twice with 3.75 ml of 0.25 M sodium bicarbonate (pH 9.3) and0.5 M NaCl followed by 0.5 ml of 50 mM sodium acetate (pH 4.5). Theproduct was eluted with 2.5 ml of 50 mM sodium acetate (pH 4.5)containing 0.1 M sorbitol and collected in scintillation vials. Theeluate was mixed with 4.5 ml Ecolite Scintillation Cocktail and theradioactivity measured by liquid scintillation spectrometry.

Both the construct designed to express the complete bovine open readingframe and that designed to express a truncated protein, in either pADNSor pADANS, expressed active protein as determined by biochemicalphosphodiesterase assay of cell extracts. Extracts of 10DAB harboringpcam61met140 yielded measurable phorphodiesterase activity (see, infra,second method of part D) while the extract of 10DAB cells harboringpcamH61-6N-7 lacked detectable activity.

C. Yeast Phenotype Complementation by Expression of a cDNA Encoding acGS-PDE

The plasmid p3cGS-5, which contains a 4.2-kb DNA fragment encoding thebovine cGS-PDE, was adapted for cloning into pADNS and pADANS byreplacing the first 147 bases of the cDNA with a restriction sitesuitable for use in insertion into plasmids. The oligonucleotide BS1,having the sequence 5′TACGAAGCTTTGATGCGCCGACAGCCTGC, SEQ ID NO:57

encodes a HindIII site and anneals to positions 148-165 of the cDNAinsert. An oligonucleotide designated BS3 GGTCTCCTGTTGCAGATATTG, SEQ IDNO:58anneals to positions 835-855 just 3′ of a unique NsiI site. Theresulting PCR-generated fragment following digestion with HindIII andNsiI was then ligated to HindIII- and NsiI-digested p3cGS-5 therebyreplacing the original 5′ end of the bovine cDNA. A plasmid derived fromthis ligation was digested with HindIII and NotI to release the modifiedcDNA insert. The insert was cloned into pADNS and pADANS at theirHindIII and NotI sites. These plasmids were then transformed into theyeast strain 10DAB by the lithium acetate method and the transformedcells were grown and subjected to elevated temperatures as in Section A,above. Yeast transformed with plasmids constructed to express the bovinecGS-PDE were resistant to the thermal pulse.

In a like manner, plasmid pHcgs6n (Example VI) was transformed intoyeast host strain YKS45 by lithium acetate transformation. Heat shockanalysis was performed as above except that the plates were initiallygrown two days at 30° C. and the warmed plates were maintained at 56° C.for 10, 20, 30 and 45 minutes. Yeast transformed with the plasmiddesigned to express the full length human cGS-PDE was resistant tothermal pulse.

D. Biochemical Assay of Expression Product

The expression of the bovine cGS-PDE was also evaluated by preparingcell-free extracts from the yeast and measuring the extracts'biochemical phosphodiesterase activity. For this purpose, 50 ml culturesof transformed 10DAB yeast cells were grown in liquid SC-leucine to adensity of about 10 million cells per ml. Sherman et al., Methods inYeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1986). The cells were collected by centrifugation, the cell pelletswere washed once with water, and the final cell pellets were frozen. Toprepare an extract, the frozen cells were thawed on ice, mixed with 1 mlof PBS and an equal volume of glass beads, vortexed to disrupt the yeastcells, and centrifuged to remove debris. The supernatant was thenassayed for phosphodiesterase activity as in Section B, above.Constructs in either pADNS or pADANS expressed active protein asdetermined by biochemical phosphodiesterase assay of cell extracts.

YKS45 transformed with plasmid pHcgs6n were grown in SC-leu medium to1-2×10⁷ cells/ml. The cells were harvested by centrifugation and thecell pellets were frozen. A frozen cell pellet, typically containing10¹⁰ cells, was mixed with lysis buffer (25 mM Tris HCl pH 8, 5 mM EDTA,5 mM EGTA, 1 mM o-phenathroline, 0.5 mM AEBSF, 0.01 mg/mL pepstatin,0.01 mg/mL leupeptin, 0.01 mg/mL aprotinin, 0.1% 2-mercaptoethanol) tobring the total volume to 2.5 ml. The mixture was thawed on ice and thenadded to an equal volume of glass beads. The cells were disrupted bycycles of vortexing and chilling on ice, then additional lysis bufferwas mixed with the disrupted cells to bring the total lysis buffer addedto 5 ml. The suspension was centrifuged for 5 min. at 12,000×g. Thesupernatant was removed and either assayed immediately or frozen rapidlyin a dry ice ethanol bath and stored at −70° C.

Phosphodiesterase activity was assayed by mixing an aliquot of cellextract in (40 mM Tris-Cl pH 8.0, 1.mM EGTA, 0.1 mg/mL BSA) containing 5mM MgCl₂ and radioactive substrate, incubating at 30° C. for up to 30min. and terminating the reaction with stop buffer (0.1M ethanolamine pH9.0, 0.5M ammonium sulfate, 10 mM EDTA, 0.05% SDS final concentration).The product was separated from the cyclic nucleotide substrate bychromatography on BioRad Affi-Gel 601. The sample was applied to acolumn containing approximately 0.25 ml of Affi-Gel 601 equilibrated incolumn buffer (0.1M ethanolamine pH 9.0 containing 0.5M ammoniumsulfate). The column was washed five times with 0.5 ml of column buffer.The product was eluted with four 0.5 ml aliquots of 0.25M acetic acidand mixed with 5 ml Ecolume (ICN Biochemicals). The radioactive productwas measured by scintillation counting. Extracts from yeast expressingthe human cGS-PDE hydrolyzed both cyclic AMP and cyclic GMP, as expectedfor this isozyme.

EXAMPLE IX Tissue Expression Studies Involving CaM-PDE and cGS-PDEPolvnucleotides

A. Northern Blot Analysis

DNAs isolated in Examples I, III, and IV above were employed to developprobes for screening total or poly A-selected RNAs isolated from avariety of tissues and the results are summarized below.

1. Northern analysis was performed on mRNA prepared from a variety ofbovine adrenal cortex, adrenal medulla, heart, aorta, cerebral cortex,basal ganglia, hippocampus, cerebellum, medulla/spinal cord, liver,kidney cortex, kidney medulla, kidney papillae, trachea, lung, spleenand T-lymphocyte tissues using an approximately 3 kb radiolabeled cDNAfragment isolated from plasmid p3cGS-5 upon digestion with EcoRI andSmaI. A single 4.5 kb mRNA species was detected in most tissues. Thesize of the cGS-PDE mRNA appeared to be slightly larger (approximately4.6 kb) in RNA isolated from cerebral cortex, basal ganglia andhippocampus. The cGS PDE mRNA was most abundant in adrenal cortex. Itwas also abundant in adrenal medulla and heart. It appeared to bedifferentially expressed in anatomically distinct regions of the brainand kidney. Among RNAs isolated from five different brain regions, cGSPDE mRNA was most abundant in hippocampus, cerebral cortex, and basalganglia. Very little cGS PDE transcript was detected in cerebellum ormedulla and spinal cord RNAs. Although the cGS PDE mRNA was detected inall regions of the kidney, it appeared to be most abundant in the outerred medulla and papillae. The cGS PDE mRNA was also detected in liver,trachea, lung, spleen, and T-lymphocyte RNA. Very little cGS PDE mRNAwas detected in RNA isolated from aorta.

2. Radiolabeled DNA probes were prepared from random hexamer primedfragments extended on heat denatured 1.6 kb EcoRI restrictionendonuclease fragments of the cDNA insert of plasmid pCAM-40. InNorthern analysis, the DNA probes hybridized with 3.8 and 4.4 kb mRNAsin brain and most of the other tissues analyzed including cerebralcortex, basal ganglia, hippocampus, cerebellum, medulla and spinal cord,heart, aorta, kidney medulla, kidney papillae, and lung. Hybridizationof probe with the 3.8 kb mRNA from liver, kidney cortex and trachea wasonly detected after longer autoradiographic exposure.

3. Northern blot analysis of mRNA from several tissues of the centralnervous system was carried out using a subcloned, labeled p12.3a DNAfragment (containing most of the conserved PDE catalytic domain) as aprobe. The most intense hybridization signal was seen in mRNA from thebasal ganglia and strong signals were also seen in mRNA from othertissues including kidney papilla and adrenal medulla.

B. RNAse Protection

1. Three antisense riboprobes were constructed. Probe III corresponds tothe catalytic domain-encoding region of p3cGS-5 (273 bp corresponding tobases 2393 through 2666 of SEQ. ID NO: 38); probe II to the cGMP-bindingdomain encoding (468 bp corresponding to bases 959 through 1426; andprobe I to the 5′ end and portions of amino terminal-encoding region(457 bases corresponding to bases 1 through 457).

Total RNAs extracted from all of the examined tissues completelyprotected probes II and III. Nearly complete protection (457 bases) ofriboprobe I with RNAs isolated from adrenal cortex, adrenal medulla, andliver was also observed. However, RNA isolated from cerebral cortex,basal ganglia, and hippocampus only protected an approximately 268-basefragment of riboprobe I. A relatively small amount of partiallyprotected probe I identical in size with the major fragments observed inthe brain RNA samples was also detected in RNAs isolated from all of theexamined tissues except liver. Interestingly, heart RNA yielded bothcompletely protected (457 base) riboprobe and, like brain RNA, a268-base fragment. Unlike the protection pattern observed using RNAsisolated from any of the other tissues, however, the partially protectedriboprobe I fragment appeared to be more abundant. The results suggestthat two different cGS-PDE RNA species are expressed.

2. Radiolabeled antisense riboprobes corresponding to a portion ofeither the CaM-binding domain on the catalytic domain of CaM-PDE wereconstructed from restriction endonuclease cleavage fragments (AccI/SstIand Tth111I/HincII) of pCaM-40cDNA. Total RNAs isolated from fivedifferent brain regions (cerebral cortex, basal ganglia, hippocampus,cerebellum, and medulla/spinal cord) completely protected the antisenseriboprobes encoding both the CaM-binding and catalytic domains. TotalRNAs from heart, aorta, lung, trachea and kidney completely protectedthe riboprobe corresponding to the catalytic domain but only protectedabout 150 bases of the CaM-binding domain riboprobe, suggesting that anisoform structurally related to the 61 kD CaM-PDE is expressed in thesetissues.

3. Antisense riboprobes were generated based on plasmid p12.3a andcorresponding to bases −1 through 363 and 883-1278 of SEQ. ID NO: 26.The former probe included 113 bases of the 5′ noncoding sequence as wellas the start methionine codon through the putative CaM-binding domain,while the latter encoded the catalytic domain. Among all tissuesassayed, RNA from basal ganglia most strongly protected each probe.Strong signals of a size corresponding to the probe representing theamino terminus were observed in protection by cerebral cortex,cerebellum, basal ganglia, hippocampus and adrenal medulla RNA. Noprotection was afforded to this probe by kidney papilla or testis RNAeven though the tissue showed signals on the Northern analysis and RNAseprotection of the conserved domain probe, suggesting that a structurallyrelated isozyme is expressed in this tissue.

While the present invention has been described in terms of specificmethods and compositions, it is understood that variations andmodifications will occur to those skilled in the art upon considerationof the invention. Consequently only such limitations as appear in theappended claims should be placed thereon. Accordingly, it is intended inthe appended claims to cover all such equivalent variations which comewithin the scope of the invention as claimed.

1. A purified and isolated polynucleotide sequence consisting essentially of polynucleotide sequence encoding a mammalian Ca²⁺/calmodulin stimulated cyclic nucleotide phosphodiesterase polypeptide.
 2. A purified and isolated polynucleotide sequence consisting essentially of polynucleotide sequence encoding a mammalian cyclic GMP stimulated cyclic nucleotide phosphodiesterase polypeptide.
 3. A polynucleotide sequence according to claim 1 or 2 which encodes a human phosphodiesterase polypeptide.
 4. A polynucleotide sequence according to claim 3 selected from the group consisting of the human DNA inserts present in vectors pGSPDE6.1 (A.T.C.C. 68583), pGSPDE7.1 (A.T.C.C. 68585), pGSPDE9.2 (A.T.C.C. 68584), λ CaM H6a (A.T.C.C. 75000) pcamH3EF (A.T.C.C. 68964), pHcam61-6N-7 (A.T.C.C. 68963), pcamhella (A.T.C.C. 68965), and pHcgs6n (A.T.C.C. 68962).
 5. A polynucleotide sequence according to claim 1 or 2 which encodes a bovine phosphodiesterase polypeptide.
 6. A polynucleotide sequence according to claim 5 which encodes a bovine brain 61 kDa Ca²⁺/calmodulin stimulated phosphodiesterase polypeptide.
 7. A polynucleotide sequence according to claim 5 encoding a bovine brain 63 kDa Ca²⁺/calmodulin stimulated phosphodiesterase polypeptide.
 8. A polynucleotide sequence according to claim 5 which encodes a bovine heart 59 kDa Ca²⁺/calmodulin stimulated phosphodiesterase polypeptide.
 9. A DNA sequence according to claim 8 which is SEQ ID NO:
 16. 10. A DNA sequence according to claim 5 selected from the group consisting of the bovine DNA inserts present in vectors p12.3A (A.T.C.C. 68577), pCaM-40 (A.T.C.C. 68576), pBBcGS PDE-5 (A.T.C.C. 68578), pBBcGS PDE-7 (A.T.C.C. 68580), and p3cGS-5 (A.T.C.C. 68579).
 11. A cDNA sequence according to claims 1 or
 2. 12. A genomic DNA sequence according to claims 1 or
 2. 13. A DNA vector, having inserted therein a DNA sequence according to claim 1 or
 2. 14. A procaryotic or eucaryotic host cell stably transformed with a polynucleotide sequence according to claim 1 or
 2. 15. A yeast host cell according to claim
 14. 16. A polypeptide product of the expression in a transformed procaryotic or eucaryotic host cell of a polynucleotide sequence according to claim 1 or
 2. 17. A polypeptide product according to claim 16 as expressed in a yeast host cell.
 18. A purified and isolated polynucleotide sequence consisting essentially of a polynucleotide sequence encoding a polypeptide having the enzymatic activity of a mammalian Ca²⁺/calmodulin stimulated cyclic nucleotide phosphodiesterase and selected from the group consisting of: (a) the mammalian DNA inserts in vectors pCaM-40 (A.T.C.C. 68576), p12.3 (A.T.C.C. 68577), and pHcam61-6N-7 (A.T.C.C. 68963); (b) polynucleotide sequences which hybridize under stringent hybridization conditions to a DNA sequence selected from the mammalian DNA inserts in vectors pCAM-40 (A.T.C.C. 68576), p12.3A (A.T.C.C. 68577), λ CaM H6a (A.T.C.C. 75000), pHcam61-6N-7 (A.T.C.C. 68963), pcamH3EF (A.T.C.C. 68964), and pcamHella (A.T.C.C. 68965); (c) polynucleotide sequences which hybridize under stringent hybridization conditions to the sequence set forth in SEQ ID NO: 16; (d) polynucleotide sequences encoding the same polypeptide as the polynucleotide sequences of (a), (b) and (c) above by means of degenerate codons.
 19. A polypeptide product of the expression in a transformed or transfected procaryotic or eucaryotic host cell of a polynucleotide sequence according to claim
 18. 20. A polypeptide product according to claim 19 as expressed in a yeast host cell.
 21. A purified and isolated polynucleotide sequence consisting essentially of a polynucleotide sequence encoding a polypeptide having the enzymatic activity of a mammalian cyclic GMP stimulated nucleotide phosphodiesterase and selected from the group consisting of: (a) the mammalian DNA inserts in vectors p3cGS-5.(A.T.C.C. 68579) and pHcgs6n (A.T.C.C. 68962); (b) polynucleotide sequences which hybridize under stringent hybridization conditions to the mammalian DNA inserts in vectors p3cGS-5 (A.T.C.C. 68579), pHcgs6n (A.T.C.C. 68962), pGSPDE6.1 (A.T.C.C. 68583), pGSPDE7.1 (A.T.C.C. 68585), pGSPDE9.2 (A.T.C.C. 68584), pBBCGSPDE-5 (A.T.C.C. 68578) and pBBCGSPDE-7 (A.T.C.C. 68580); and (c) DNA sequences encoding the same polypeptide as the DNA sequences of (a) and (b) above by means of degenerate codons.
 22. A polypeptide product of the expression in a transformed or transfected procaryotic or eucaryotic host cell of a polynucleotide sequence according to claim
 21. 23. A polypeptide product according to claim 22 as expressed in a yeast host cell.
 24. An antibody substance specifically immunoreactive with a polypeptide product according to claim 16, 19 or
 22. 25. A method for producing a polypeptide having the enzymatic activity of a mammalian Ca²⁺/calmodulin stimulated cyclic nucleotide phosphodiesterase, said method comprising: (a) stably transforming or transfecting a procaryotic or eucaryotic host cell with a polynucleotide sequence according to claim 1 or 18; and (b) growing the host cell formed in step (a) in a nutrient medium under conditions allowing expression of said DNA sequence in said host cell.
 26. A method according to claim 25 further including the step of isolating the polypeptide product of expression of said polynucleotide sequence in said host cell.
 27. A method according to claim 25 wherein said host cell is a yeast host cell.
 28. A method for producing a polypeptide having the enzymatic activity of a cyclic GMP stimulated cyclic nucleotide phosphodiesterase, said method comprising: (a) stably transforming or transfecting a procaryotic or eucaryotic host cell with a polynucleotide sequence according to claim 2 or 21; and (b) growing the host cell formed in step (a) in a nutrient medium under conditions allowing expression of said DNA sequence in said host cell.
 29. A method according to claim 28 further including the step of isolating the polypeptide product of expression of said polynucleotide sequence in said host cell.
 30. A method according to claim 28 wherein said host cell is a yeast host cell.
 31. An assay method for identifying a chemical agent which modifies the enzymatic activity of a mammalian Ca²⁺/calmodulin sensitive cyclic nucleotide phosphodiesterase, said method comprising: (a) stably transforming, with a polynucleotide sequence according to claim 1 or 18, a procaryotic or eucaryotic host cell having a phenotypic character susceptible to alteration upon expression of said polynucleotide sequence; (b) growing the host cell formed in step (a) in a nutrient medium under conditions allowing expression of said polynucleotide sequence in said host cell accompanied by the corresponding alteration in the host cell phenotype; (c) contacting the host cells grown according to step (b) with a chemical agent to be assayed; and, (d) determining any modification in the alteration of the phenotype of said host cells contacted with said chemical agent in step (c).
 32. An assay method according to claim 31 wherein said host cell is a yeast host cell.
 33. An assay method for identifying a chemical agent which modifies the enzymatic activity of a mammalian cyclic GMP stimulated cyclic nucleotide phosphodiesterase, said method comprising: (a) stably transforming, with a polynucleotide sequence according to claim 2 or 21, a procaryotic or eucaryotic host cell having a phenotypic character susceptible to alteration upon expression of said polynucleotide sequence in said host; (b) growing the host cell formed in step (a) in a nutrient medium under conditions allowing expression of said polynucleotide sequence in said host cell accompanied by the corresponding alteration in the host cell phenotype; (c) contacting the host cells grown according to step (b) with a chemical agent to be assayed; and, (d) determining any modification in the alteration of the phenotype of said host cells contacted with said chemical agent in step (d).
 34. An assay method according to claim 33 wherein the host cell is a yeast host cell.
 35. An assay method for identifying a chemical agent which modifies the enzymatic activity of a mammalian Ca²⁺/calmodulin sensitive cyclic nucleotide phosphodiesterase, said method comprising: (a) stably transforming, with a polynucleotide sequence according to claim 1 or 18, a procaryotic or eucaryotic host cell having a phenotypic character susceptible to alteration upon expression of said polynucleotide sequence; (b) growing the host cell formed in step (a) in a nutrient medium under conditions allowing expression of said polynucleotide sequence in said host cell accompanied by the corresponding alteration in the host cell phenotype; (c) identifying said host cells having an altered phenotype; (d) disrupting said host cell; (e) isolating cytosol from said disrupted host cell; (f) contacting said cytosol with said chemical agent; and (g) determining whether said enzymatic activity has been altered.
 36. An assay method according to claim 35 wherein said host cell is a yeast host cell.
 37. An assay method for identifying a chemical agent which modifies the enzymatic activity of a mammalian cyclic GMP stimulated cyclic nucleotide phosphodiesterase, said method comprising: (a) stably transforming, with a polynucleotide sequence according to claim 2 or 21, a procaryotic or eucaryotic host cell having a phenotypic character susceptible to alteration upon expression of said polynucleotide sequence in said host; (b) growing the host cell formed in step (a) in a nutrient medium under conditions allowing expression of said polynucleotide sequence in said host cell phenotype; (c) identifying said host cells having an altered phenotype; (d) disrupting said host cell; (e) isolating cytosol from said disrupted host cell; (f) contacting said cytosol with said chemical agent; and (g) determining whether said enzymatic activity has been altered.
 38. An assay method according to claim 37 wherein the host cell is a yeast host cell. 